Method and system of identifying and quantifying a protein

ABSTRACT

Methods and system for identifying and/or quantifying a protein are provided herein.

FIELD

The invention generally pertains to a method and system of identifyingand/or quantifying a protein.

BACKGROUND

Protein based biopharmaceutical products must meet very high standardsof purity. There are several process-related impurities andproduct-related impurities that are found in biopharmaceuticals. Theseimpurities do not have properties comparable to those of the desiredproduct with respect to activity, efficacy, and safety. One example ispost-translational modifications (PTMs) of the protein which profoundlyaffect protein properties relevant to their therapeutic application.Another such example includes the homodimeric contaminants that can bepresent during the production of bispecific antibody which ideally mustbe removed by downstream purification. These impurities could exhibit adifferent mode of action and potential toxicity or immunogenicitycompared to the product. In addition, they can have a lower stabilitythan the product which presents a higher risk for aggregation andimmunogenicity. Despite the recent advances, the challenge to developpurity assay methods for quantitative evaluation of such impuritiesremains. Additionally, a key challenge in analytical method developmentfor bispecific antibodies can be that the method must accurately andreproducibly detect impurities present at 2% or lower level relative tothe main desired species. Therefore, it is important to monitor andcharacterize such impurities during different stages of drug developmentand production. Despite the importance of impurities for biologicalfunction, their study on a large scale has been hampered by a lack ofsuitable methods.

Analytical methods for purity assays must display sufficient accuracyand resolution to detect and quantify desired product and theirimpurities. Evaluation of impurities, such as PTMs in antibodies andhomodimers in bispecific antibodies, can be difficult due tosimilarities between structural and physicochemical properties of suchimpurities and the desired product. Direct analysis of such impuritiesrequires isolation of the desired product in a sufficiently large amountfor the assay which is undesirable and only been possible in selectedcases.

Thus, there is a long felt need in the art for a method and/or systemfor identifying and quantifying a protein—impurities and/or the desiredproduct in a protein based biopharmaceuticals.

SUMMARY

Growth in the development, manufacture and sale of protein-basedbiopharmaceutical products has led to an increasing demand for methodand/or system for identification and quantification of impurities in theproducts.

Embodiments disclosed herein satisfy the aforementioned demands byproviding methods and systems for the rapid characterization ofproteins.

The disclosure, at least in part, provides a method for quantifying animpurity in a sample.

In one exemplary embodiment, the method can comprise contacting thesample to a chromatographic system having a mixed-mode size exclusionchromatography resin with an additional functionality, washing themixed-mode size exclusion chromatography resin using a mobile phase toprovide an eluent including the impurity, and quantifying an amount ofthe impurity in the eluent using a mass spectrometer.

In one aspect of this embodiment, the method for quantifying an impurityin a sample can comprise contacting said sample to a chromatographicsystem having a mixed-mode size exclusion chromatography resin with ahydrophobic interaction functionality

In one aspect of this embodiment, the method for quantifying an impurityin a sample can comprise contacting said sample to a chromatographicsystem having a mixed-mode size exclusion chromatography resin with acharge-charge interaction functionality.

In one aspect of this embodiment, the method for quantifying an impurityin a sample can comprise contacting about 10 μg to about 100 μg of asample to a chromatographic system having a mixed-mode size exclusionchromatography resin with an additional functionality.

In one aspect of this embodiment, the method for quantifying an impurityin a sample can comprise washing the mixed-mode size exclusionchromatography resin using a mobile phase to provide an eluent includingthe impurity.

In one aspect of this embodiment, the method for quantifying an impurityin a sample can comprise washing the mixed-mode size exclusionchromatography resin using a mobile phase that can be compatible with amass spectrometer.

In one aspect of this embodiment, the method for quantifying an impurityin a sample can comprise washing the mixed-mode size exclusionchromatography resin using a mobile phase, wherein the mobile phase canbe selected from ammonium acetate, ammonium bicarbonate, or ammoniumformate, or combinations thereof.

In one aspect of this embodiment, the method for quantifying an impurityin a sample can comprise washing the mixed-mode size exclusionchromatography resin using a mobile phase containing up to 600 mM totalsalt concentration.

In one aspect of this embodiment, the method for quantifying an impurityin a sample can comprise washing the mixed-mode size exclusionchromatography resin using a mobile phase with a flow rate of 0.2 ml/minto 0.4 ml/min.

In one aspect of this embodiment, the method for quantifying an impuritycan comprise contacting the sample to a chromatographic system having amixed-mode size exclusion chromatography resin with an additionalfunctionality, wherein the impurity can be a product-related impurity.

In one aspect of this embodiment, the method for quantifying an impuritycan comprise contacting the sample to a chromatographic system having amixed-mode size exclusion chromatography resin with an additionalfunctionality, wherein the impurity can be a process-related impurity.

In one aspect of this embodiment, the method for quantifying an impuritycan comprise contacting the sample to a chromatographic system having amixed-mode size exclusion chromatography resin with an additionalfunctionality, wherein the impurity can be a degradation product of aprotein.

In one aspect of this embodiment, the method for quantifying an impuritycan comprise contacting the sample to a chromatographic system having amixed-mode size exclusion chromatography resin with an additionalfunctionality, wherein the impurity can be a digestion product of aprotein.

In one aspect of this embodiment, the method for quantifying an impuritycan comprise contacting the sample to a chromatographic system having amixed-mode size exclusion chromatography resin with an additionalfunctionality, wherein the impurity can be a homodimer species of amultispecific antibody product.

In one aspect of this embodiment, the method for quantifying an impuritycan comprise contacting the sample to a chromatographic system having amixed-mode size exclusion chromatography resin with an additionalfunctionality, wherein the impurity can be a post-translationalmodification of a protein.

In one aspect of this embodiment, the method for quantifying an impuritycan comprise quantifying an amount of the impurity in said eluent usinga mass spectrometer, wherein the mass spectrometer can be a tandem massspectrometer.

In one aspect of this embodiment, the method for quantifying an impuritycan comprise quantifying an amount of the impurity in said eluent usinga mass spectrometer, wherein the mass spectrometer can be a native massspectrometer.

This disclosure, at least in part, provides a method for detecting animpurity in a sample.

In one exemplary embodiment, the method can comprise contacting thesample to a chromatographic system having a mixed-mode size exclusionchromatography resin with an additional functionality, washing themixed-mode size exclusion chromatography resin using a mobile phase toprovide an eluent including the impurity, and quantifying an amount ofthe impurity in the eluent using a mass spectrometer.

In one aspect of this embodiment, the method for detecting an impurityin a sample can comprise contacting said sample to a chromatographicsystem having a mixed-mode size exclusion chromatography resin with ahydrophobic interaction functionality

In one aspect of this embodiment, the method for detecting an impurityin a sample can comprise contacting said sample to a chromatographicsystem having a mixed-mode size exclusion chromatography resin with acharge-charge interaction functionality.

In one aspect of this embodiment, the method for detecting an impurityin a sample can comprise contacting about 10 μg to about 100 μg of asample to a chromatographic system having a mixed-mode size exclusionchromatography resin with an additional functionality.

In one aspect of this embodiment, the method for detecting an impurityin a sample can comprise washing the mixed-mode size exclusionchromatography resin using a mobile phase to provide an eluent includingthe impurity.

In one aspect of this embodiment, the method for detecting an impurityin a sample can comprise washing the mixed-mode size exclusionchromatography resin using a mobile phase that can be compatible with amass spectrometer.

In some specific exemplary embodiments, the method for detecting animpurity in a sample can comprise washing the mixed-mode size exclusionchromatography resin using a mobile phase, wherein the mobile phase canbe selected from ammonium acetate, ammonium bicarbonate, or ammoniumformate, or combinations thereof.

In some specific exemplary embodiments, the method for detecting animpurity in a sample can comprise washing the mixed-mode size exclusionchromatography resin using a mobile phase containing up to 600 mM totalsalt concentration.

In one aspect of this embodiment, the method for detecting an impurityin a sample can comprise washing the mixed-mode size exclusionchromatography resin using a mobile phase with a flow rate of 0.2 ml/minto 0.4 ml/min.

In one aspect of this embodiment, the method for detecting an impuritycan comprise contacting the sample to a chromatographic system having amixed-mode size exclusion chromatography resin with an additionalfunctionality, wherein the impurity can be a product-related impurity.

In one aspect of this embodiment, the method for detecting an impuritycan comprise contacting the sample to a chromatographic system having amixed-mode size exclusion chromatography resin with an additionalfunctionality, wherein the impurity can be a process-related impurity.

In one aspect of this embodiment, the method for detecting an impuritycan comprise contacting the sample to a chromatographic system having amixed-mode size exclusion chromatography resin with an additionalfunctionality, wherein the impurity can be a degradation product of aprotein.

In one aspect of this embodiment, the method for detecting an impuritycan comprise contacting the sample to a chromatographic system having amixed-mode size exclusion chromatography resin with an additionalfunctionality, wherein the impurity can be a digestion product of aprotein.

In one aspect of this embodiment, the method for detecting an impuritycan comprise contacting the sample to a chromatographic system having amixed-mode size exclusion chromatography resin with an additionalfunctionality, wherein the impurity can be a homodimer species of amultispecific antibody product.

In one aspect of this embodiment, the method for detecting an impuritycan comprise contacting the sample to a chromatographic system having amixed-mode size exclusion chromatography resin with an additionalfunctionality, wherein the impurity can be a post-translationalmodification of a protein.

In one aspect of this embodiment, the method for detecting an impuritycan comprise detecting an amount of the impurity in eluent using a massspectrometer, wherein the mass spectrometer can be a tandem massspectrometer.

In one aspect of this embodiment, the method for detecting an impuritycan comprise detecting an amount of the impurity in said eluent using amass spectrometer, wherein the mass spectrometer can be a native massspectrometer.

This disclosure, at least in part, provides a method for detectingand/or quantifying a target protein in a sample.

In one exemplary embodiment, the method can comprise contacting thesample to a chromatographic system having a mixed-mode size exclusionchromatography resin with an additional functionality, washing themixed-mode size exclusion chromatography resin using a mobile phase toprovide an eluent including the target protein, and detecting and/orquantifying an amount of the target protein in the eluent using a massspectrometer.

In one aspect of this embodiment, the method for detecting and/orquantifying a target protein in a sample can comprise contacting saidsample to a chromatographic system having a mixed-mode size exclusionchromatography resin with a hydrophobic interaction functionality

In one aspect of this embodiment, the method for detecting and/orquantifying a target protein in a sample can comprise contacting saidsample to a chromatographic system having a mixed-mode size exclusionchromatography resin with a charge-charge interaction functionality.

In one aspect of this embodiment, the method for detecting and/orquantifying a target protein in a sample can comprise contacting about10 μg to about 100 μg of a sample to a chromatographic system having amixed-mode size exclusion chromatography resin with an additionalfunctionality.

In one aspect of this embodiment, the method for detecting and/orquantifying a target protein in a sample can comprise washing themixed-mode size exclusion chromatography resin using a mobile phase toprovide an eluent including the impurity.

In one aspect of this embodiment, the method for detecting and/orquantifying a target protein in a sample can comprise washing themixed-mode size exclusion chromatography resin using a mobile phase thatcan be compatible with a mass spectrometer. In a specific aspect, themethod for detecting and/or quantifying a target protein in a sample cancomprise washing the mixed-mode size exclusion chromatography resinusing a mobile phase, wherein the mobile phase can be selected fromammonium acetate, ammonium bicarbonate, or ammonium formate, orcombinations thereof. In another specific aspect, the method fordetecting and/or quantifying a target protein in a sample can comprisewashing the mixed-mode size exclusion chromatography resin using amobile phase containing up to 600 mM total salt concentration.

In one aspect of this embodiment, the method for detecting and/orquantifying a target protein in a sample can comprise washing themixed-mode size exclusion chromatography resin using a mobile phase witha flow rate of 0.2 ml/min to 0.4 ml/min.

In one aspect of this embodiment, the method for detecting and/orquantifying a target protein can comprise contacting the sample to achromatographic system having a mixed-mode size exclusion chromatographyresin with an additional functionality, wherein the target protein canbe an antibody.

In one aspect of this embodiment, the method for detecting and/orquantifying a target protein can comprise contacting the sample to achromatographic system having a mixed-mode size exclusion chromatographyresin with an additional functionality, wherein the target protein canbe a bispecific antibody.

In one aspect of this embodiment, the method for detecting and/orquantifying a target protein can comprise contacting the sample to achromatographic system having a mixed-mode size exclusion chromatographyresin with an additional functionality, wherein the target protein canbe a therapeutic protein.

In one aspect of this embodiment, the method for detecting and/orquantifying a target protein can comprise contacting the sample to achromatographic system having a mixed-mode size exclusion chromatographyresin with an additional functionality, wherein the target protein canbe an impurity.

In one aspect of this embodiment, the method for detecting and/orquantifying a target protein can comprise contacting the sample to achromatographic system having a mixed-mode size exclusion chromatographyresin with an additional functionality, wherein the target protein canbe a process-related impurity of a biopharmaceutical process ofmanufacturing a protein.

In one aspect of this embodiment, the method for detecting and/orquantifying a target protein can comprise contacting the sample to achromatographic system having a mixed-mode size exclusion chromatographyresin with an additional functionality, wherein the target protein canbe a product-related impurity of a biopharmaceutical process ofmanufacturing a protein.

In one aspect of this embodiment, the method for detecting and/orquantifying a target protein can comprise contacting the sample to achromatographic system having a mixed-mode size exclusion chromatographyresin with an additional functionality, wherein the target protein canbe a degradation product of a protein.

In one aspect of this embodiment, the method for detecting and/orquantifying a target protein can comprise contacting the sample to achromatographic system having a mixed-mode size exclusion chromatographyresin with an additional functionality, wherein the target protein canbe a digestion product of a protein.

In one aspect of this embodiment, the method for detecting and/orquantifying a target protein can comprise contacting the sample to achromatographic system having a mixed-mode size exclusion chromatographyresin with an additional functionality, wherein the target protein canbe a homodimer species of a multispecific antibody product.

In one aspect of this embodiment, the method for detecting and/orquantifying a target protein can comprise quantifying an amount of thetarget protein in said eluent using a mass spectrometer, wherein themass spectrometer can be a tandem mass spectrometer.

In one aspect of this embodiment, the method for detecting and/orquantifying a target protein can comprise quantifying an amount of thetarget protein in said eluent using a mass spectrometer, wherein themass spectrometer can be a native mass spectrometer.

In one exemplary embodiment, this disclosure, at least in part, providesa mixed-mode chromatographic system a chromatographic column having amixed-mode size exclusion chromatography resin with an additionalfunctionality and a mass spectrometer.

In one aspect of this embodiment, the mixed-mode chromatographic systemcan comprise a mixed-mode size exclusion chromatography resin withhydrophobic interaction functionality.

In one aspect of this embodiment, the mixed-mode chromatographic systemcan comprise a mixed-mode size exclusion chromatography resin withcharge-charge interaction functionality.

In one aspect of this embodiment, the mixed-mode chromatographic systemcan comprise a mixed-mode size exclusion chromatography resin with anadditional functionality which can be used for elution of about 10 μg toabout 100 μg of a sample.

In one aspect of this embodiment, the mixed-mode chromatographic systemcan comprise a mixed-mode size exclusion chromatography resin capable ofreceiving a mobile phase.

In one aspect of this embodiment, the mixed-mode chromatographic systemcan comprise a mixed-mode size exclusion chromatography resin furthercapable of receiving a sample having a target protein.

In one aspect of this embodiment, the mixed-mode chromatographic systemcan comprise a mixed-mode size exclusion chromatography resin capable ofbeing washed with a mobile phase.

In one aspect of this embodiment, the mixed-mode chromatographic systemcan comprise a mass spectrometer coupled to a chromatographic columnhaving a mixed-mode size exclusion chromatography resin with anadditional functionality.

In one aspect of this embodiment, the mixed-mode chromatographic systemcan comprise a tandem mass spectrometer.

In one aspect of this embodiment, the mixed-mode chromatographic systemcan comprise a native spectrometer.

In one aspect of this embodiment, the mixed-mode chromatographic systemcan comprise a chromatographic column having a mixed-mode size exclusionchromatography resin with an additional functionality, wherein themixed-mode size exclusion chromatography resin can be compatible with amobile phase selected from ammonium acetate, ammonium bicarbonate, orammonium formate, or combinations thereof.

In one aspect of this embodiment, the mixed-mode chromatographic systemcan comprise a chromatographic column having a mixed-mode size exclusionchromatography resin with an additional functionality, wherein themixed-mode size exclusion chromatography resin can be washed using amobile phase containing up to 600 mM total salt concentration.

In one aspect of this embodiment, the mixed-mode chromatographic systemcan comprise a chromatographic column having a mixed-mode size exclusionchromatography resin with an additional functionality, wherein thechromatographic column can be washed with a mobile phase with a flowrate of 0.2 ml/min to 0.4 ml/min.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows represents an example of a system used for quantifyingand/or detecting a protein using size exclusion chromatography or ionexchange chromatography.

FIG. 2 represents an attempt for purifying a bispecific antibody fromhomodimer species using an exemplary embodiment.

FIG. 3 shows the Hofmeister series showing the effect of anions andcations on protein precipitation (or promoting hydrophobic interaction).

FIG. 4 shows a mixed-mode size exclusion chromatography massspectrometry system according to an exemplary embodiment.

FIG. 5 shows the plots of the retention time of eight mAbs in the samplemixture on the BEH200 SEC column performed under mobile phaseconcentrations ranging from 30 mM to 300 mM, wherein the two insetsrepresent the base peak chromatograms (BPCs) from the MM-SEC-MS analysisof the eight mAbs at the corresponding concentrations according to anexemplary embodiment.

FIG. 6 shows the chromatographic profile of the bispecific antibodysample using an exemplary mixed-mode size chromatography massspectrometry system using mobile phases: 150 mM total salt concentrationand 450 mM total salt concentration.

FIG. 7 shows the extracted ion chromatogram (XIC) and the native massspectra of the bispecific antibody, homodimer 1 and homodimer 2separated and analyzed using a mixed-mode size exclusion chromatographymass spectrometry according to an exemplary embodiment.

FIG. 8 shows comparison of the total ion chromatogram (TIC) and thenative MS spectra for RP LC-MS on Lumos, SEC-MS on EMR, and MM-SEC-MS onEMR of an antibody detected spectrometry according to an exemplaryembodiment.

FIG. 9 shows extracted ion chromatograms (XIC) and the native massspectra of consecutive runs of MM-SEC-MS detection of an antibody usingZenix SEC-300, 3 μm, 300 Å, 7.8×300 mm according to an exemplaryembodiment.

FIG. 10 shows extracted ion chromatograms (XIC) obtained on performingdetection of homodimer species in a bispecific antibody productaccording to an exemplary embodiment.

FIG. 11 shows comparison of the extracted ion chromatograms (XIC)obtained on performing detection of homodimer species in a bispecificantibody product using a Zenix SEC-300, 3 μm, 300 Å, 7.8×300 mm at 0.4mL/min flow rate and Zenix SEC-300, 3 μm, 300 Å, 4.6×300 mm at 0.3mL/min flow rate according to an exemplary embodiment.

FIG. 12 shows the extracted ion chromatograms (XIC) obtained onperforming MM-SEC-MS analysis of deglycosylated mixture of bispecificantibody, homodimer 1, and homodimer 2 using mobile phase with differentsalt concentration according to an exemplary embodiment.

FIG. 13 shows the chart of retention time (minute) of a protein vs.total salt concentration of the mobile phase for a deglycosylatedmixture of bispecific antibody, homodimer 1, and homodimer 2 onperforming MM-SEC-MS analysis according to an exemplary embodiment.

FIG. 14 represents a chart showing trend in retention time based onchanging total salt concentration on performing MM-SEC-MS analysisaccording to an exemplary embodiment.

FIG. 15 represents a chart showing a trend in difference in retentiontime on changing total salt concentration on performing MM-SEC-MSanalysis according to an exemplary embodiment.

FIG. 16 shows the extracted ion chromatograms (XIC) obtained onconducting MM-SEC-MS analysis of deglycosylated mixture of bispecificantibody, homodimer 1, and homodimer 2 on Waters BEH SEC Columnaccording to an exemplary embodiment.

FIG. 17 shows the chart of retention time (minutes) of a protein vs.total salt concentration of the mobile phase for a deglycosylatedmixture of bispecific antibody, homodimer 1, and homodimer 2 onperforming MM-SEC-MS analysis on Waters BEH SEC Column according to anexemplary embodiment.

FIG. 18 shows the extracted ion chromatograms (XIC) obtained onconducting MM-SEC-MS analysis of an antibody and its oxidized variant onWaters BEH SEC Column according to an exemplary embodiment.

FIG. 19 shows the chart of retention time (minutes) of a protein vs.total salt concentration of the mobile phase for an antibody and itsoxidized variant on performing MM-SEC-MS analysis on Waters BEH SECColumn according to an exemplary embodiment.

FIG. 20 shows a method of sample preparation of the mixture containingbispecific antibody and its homodimer species according to an exemplaryembodiment.

FIG. 21 shows MM-SEC-MS analysis of mixture at intact level ofbispecific antibody and its homodimer species using mobile phase with300 mM salt concentration according to an exemplary embodiment.

FIG. 22 shows MM-SEC-MS analysis of mixture at subunit level ofbispecific antibody and its homodimer species using mobile phase with 70mM salt concentration according to an exemplary embodiment.

FIG. 23 shows the homodimer quantitation results from MM-SEC-MS analysisat intact level of bispecific antibody and its homodimer speciesaccording to an exemplary embodiment.

FIG. 24 shows the homodimer quantitation results from MM-SEC-MS analysisat subunit level of bispecific antibody and its homodimer speciesaccording to an exemplary embodiment.

FIG. 25 shows analysis of bispecific antibody mixtures [four differentbsAb/H2L2 homodimer/H*2L2 homodimer mixtures] by MM-SEC-MS on either theBEH column (left) or the Zenix column (right) carried out according toexemplary embodiments, wherein each BPC trace represents one individualanalysis using the mobile phase salt concentration indicated.

FIG. 26 shows the limit of detection study by MM-SEC-MS for thehomodimer impurities in bsAb2 (left panel) and bsAb4 (right panel) using0.01% and 0.1% spiked-in standards, respectively for analysis carriedout according to exemplary embodiments, wherein the native MS spectra(a, b, c, d, e and f) were averaged across the corresponding TIC regionsand the two most abundant charge states were shown.

FIG. 27 shows the quantitation study carried out according to exemplaryembodiments using homodimers spiked into bsAb2 at serially diluted,known ratios (grey line and the marked values on the left panel) andalso shown are the measured ratios of H2L2 homodimer (red) and H*2L2homodimer (blue) to bsAb2 based on the XIC intensity of the four mostabundant charge states in the raw mass spectrum (0.1% relative abundanceof each homodimer, as an example shown on the right panel).

DETAILED DESCRIPTION

Impurities in biopharmaceuticals can cause changes that couldpotentially impact the efficacy, clearance, safety, and immunogenicityof the desired product. For example, oxidation of methionine andtryptophan side chains can affect antibody binding to Fc receptors andantigens (Bertolotti-Ciarlet et al. Mol. Immunol. (2009) 46: 1878-1882;Pan et al. Protein Sci. (2009) 18: 424-433; Wei et al. Anal. Chem.(2007) 79: 2797-2805; and Wang et al. Mol. Immunol. (2011) 48: 860-866).

Traditional separation-based antibody purity assays such aselectrophoresis- and high-performance liquid chromatography (HPLC)-basedmethods lack the resolution needed to distinguish these impurities fromthe desired product. Peptide mapping via reverse phase liquidchromatography (RPLC) coupled with mass spectrometry used to monitorPTMs has some limitations as the sample preparation process for RP-LC-MSis lengthy, and in some cases the chromatographic conditions such ashigh temperature, organic solvents, and acidic pH could induce oxidationartifacts.

Additionally, some size exclusion chromatography or ion exchangechromatography methods can also be used for separating impurities fromthe desired product. The separated impurities and the desired productcan further be analyzed using a mass spectrometer. However, the mobilephase from the size exclusion chromatography or ion exchangechromatography column cannot be directly injected into the massspectrometer and requires additional steps including a change in themobile phase (See FIG. 1).

Considering the limitations of existing methods, an effective andefficient method for identification and quantification of impuritiesusing a novel mixed-mode—size exclusion chromatography—mass spectrometrysystem was developed as disclosed herein. The mixed-mode—size exclusionchromatography—mass spectrometry system improves the sensitivity andability to quantify impurities present at very low levels due toefficient mixed-mode separation and sensitive online MS detection whichcannot be achieved by other typical assays.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing, particular methods and materials are nowdescribed. All publications mentioned are hereby incorporated byreference.

The term “a” should be understood to mean “at least one”; and the terms“about” and “approximately” should be understood to permit standardvariation as would be understood by those of ordinary skill in the art;and where ranges are provided, endpoints are included.

Target Protein

Biopharmaceutical products are required to show high levels of potency,purity, and low level of structural heterogeneity. Structuralheterogeneity often affects the bioactivity and efficacy of a drug.Therefore, characterizing and quantifying the therapeutic protein and/orthe impurities is important in pharmaceutical drug development.Structural heterogeneity in a protein can arise from post-translationalmodifications as well as inherent chemical modifications duringmanufacturing and storage conditions. For proteins produced in thebiotechnology industry, complementary separation techniques can benecessary both to purify the target protein and to give an accuratepicture of the quality of the final product. The complexity of theproduct eliminates the use of simple one-dimensional separationstrategies. Therefore, an accurate and efficient method of detectingand/or quantifying the therapeutic protein and/or impurities is needed.

In some exemplary embodiments, the disclosure provides a method forquantifying and/or detecting a protein and/or an impurity in a sample.

As used herein, the term “protein” includes any amino acid polymerhaving covalently linked amide bonds. Proteins comprise one or moreamino acid polymer chains, generally known in the art as “polypeptides”.“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. “Synthetic peptides orpolypeptides' refers to a non-naturally occurring peptide orpolypeptide. Synthetic peptides or polypeptides can be synthesized, forexample, using an automated polypeptide synthesizer. Various solid phasepeptide synthesis methods are known to those of skill in the art. Aprotein may contain one or multiple polypeptides to form a singlefunctioning biomolecule. A protein can include any of bio-therapeuticproteins, recombinant proteins used in research or therapy, trapproteins and other chimeric receptor Fc-fusion proteins, chimericproteins, antibodies, monoclonal antibodies, polyclonal antibodies,human antibodies, and bispecific antibodies. In another exemplaryaspect, a protein can include antibody fragments, nanobodies,recombinant antibody chimeras, cytokines, chemokines, peptide hormones,and the like. Proteins may be produced using recombinant cell-basedproduction systems, such as the insect bacculovirus system, yeastsystems (e.g., Pichia sp.), mammalian systems (e.g., CHO cells and CHOderivatives like CHO-K1 cells). For a recent review discussingbiotherapeutic proteins and their production, see Ghaderi et al.,“Production platforms for biotherapeutic glycoproteins. Occurrence,impact, and challenges of non-human sialylation,” (Biotechnol. Genet.Eng. Rev. (2012) 147-75). In some embodiments, proteins comprisemodifications, adducts, and other covalently linked moieties. Thosemodifications, adducts and moieties include for example avidin,streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose,neuraminic acid, N-acetylglucosamine, fucose, mannose, and othermonosaccharides), PEG, polyhistidine, FLAGtag, maltose binding protein(MBP), chitin binding protein (CBP), glutathione-S-transferase (GST)myc-epitope, fluorescent labels and other dyes, and the like. Proteinscan be classified on the basis of compositions and solubility and canthus include simple proteins, such as, globular proteins and fibrousproteins; conjugated proteins, such as, nucleoproteins, glycoproteins,mucoproteins, chromoproteins, phosphoproteins, metalloproteins, andlipoproteins; and derived proteins, such as, primary derived proteinsand secondary derived proteins.

In some exemplary embodiments, the protein can be an antibody, abispecific antibody, a multispecific antibody, antibody fragment,monoclonal antibody, or combinations thereof.

The term “antibody,” as used herein includes immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds, as well as multimersthereof (e.g., IgM). Each heavy chain comprises a heavy chain variableregion (abbreviated herein as HCVR or V_(H)) and a heavy chain constantregion. The heavy chain constant region comprises three domains, C_(H)1,C_(H)2 and C_(H)3. Each light chain comprises a light chain variableregion (abbreviated herein as LCVR or V_(L)) and a light chain constantregion. The light chain constant region comprises one domain (C_(L)1).The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments ofthe invention, the FRs of the anti-big-ET-1 antibody (or antigen-bindingportion thereof) may be identical to the human germline sequences, ormay be naturally or artificially modified. An amino acid consensussequence may be defined based on a side-by-side analysis of two or moreCDRs. The term “antibody,” as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

As used herein, an “antibody fragment” includes a portion of an intactantibody, such as, for example, the antigen-binding or variable regionof an antibody. Examples of antibody fragments include, but are notlimited to, a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a scFvfragment, a Fv fragment, a dsFv diabody, a dAb fragment, a Fd′ fragment,a Fd fragment, and an isolated complementarity determining region (CDR)region, as well as triabodies, tetrabodies, linear antibodies,single-chain antibody molecules, and multi specific antibodies formedfrom antibody fragments. Fv fragments are the combination of thevariable regions of the immunoglobulin heavy and light chains, and ScFvproteins are recombinant single chain polypeptide molecules in whichimmunoglobulin light and heavy chain variable regions are connected by apeptide linker. In some exemplary embodiments, an antibody fragmentcontains sufficient amino acid sequence of the parent antibody of whichit is a fragment that it binds to the same antigen as does the parentantibody; in some exemplary embodiments, a fragment binds to the antigenwith a comparable affinity to that of the parent antibody and/orcompetes with the parent antibody for binding to the antigen. Anantibody fragment may be produced by any means. For example, an antibodyfragment may be enzymatically or chemically produced by fragmentation ofan intact antibody and/or it may be recombinantly produced from a geneencoding the partial antibody sequence. Alternatively or additionally,an antibody fragment may be wholly or partially synthetically produced.An antibody fragment may optionally comprise a single chain antibodyfragment. Alternatively or additionally, an antibody fragment maycomprise multiple chains that are linked together, for example, bydisulfide linkages. An antibody fragment may optionally comprise amulti-molecular complex. A functional antibody fragment typicallycomprises at least about 50 amino acids and more typically comprises atleast about 200 amino acids.

The phrase “bispecific antibody” includes an antibody capable ofselectively binding two or more epitopes. Bispecific antibodiesgenerally comprise two different heavy chains, with each heavy chainspecifically binding a different epitope—either on two differentmolecules (e.g., antigens) or on the same molecule (e.g., on the sameantigen). If a bispecific antibody is capable of selectively binding twodifferent epitopes (a first epitope and a second epitope), the affinityof the first heavy chain for the first epitope will generally be atleast one to two or three or four orders of magnitude lower than theaffinity of the first heavy chain for the second epitope, and viceversa. The epitopes recognized by the bispecific antibody can be on thesame or a different target (e.g., on the same or a different protein).Bispecific antibodies can be made, for example, by combining heavychains that recognize different epitopes of the same antigen. Forexample, nucleic acid sequences encoding heavy chain variable sequencesthat recognize different epitopes of the same antigen can be fused tonucleic acid sequences encoding different heavy chain constant regions,and such sequences can be expressed in a cell that expresses animmunoglobulin light chain. A typical bispecific antibody has two heavychains each having three heavy chain CDRs, followed by a C_(H)1 domain,a hinge, a C_(H)2 domain, and a C_(H)3 domain, and an immunoglobulinlight chain that either does not confer antigen-binding specificity butthat can associate with each heavy chain, or that can associate witheach heavy chain and that can bind one or more of the epitopes bound bythe heavy chain antigen-binding regions, or that can associate with eachheavy chain and enable binding or one or both of the heavy chains to oneor both epitopes. BsAbs can be divided into two major classes, thosebearing an Fc region (IgG-like) and those lacking an Fc region, thelatter normally being smaller than the IgG and IgG-like bispecificmolecules comprising an Fc. The IgG-like bsAbs can have differentformats, such as, but not limited to triomab, knobs into holes IgG (kihIgG), crossMab, orth-Fab IgG, Dual-variable domains Ig (DVD-Ig),Two-in-one or dual action Fab (DAF), IgG-single-chain Fv (IgG-scFv), orκλ-bodies. The non-IgG-like different formats include Tandem scFvs,Diabody format, Single-chain diabody, tandem diabodies (TandAbs),Dual-affinity retargeting molecule (DART), DART-Fc, nanobodies, orantibodies produced by the dock-and-lock (DNL) method. Fan et al. andKontermann and Brinkmann present a detailed review on bispecificantibody (Fan et al. “Bispecific antibodies and their applications” J.Hematol. Oncol. (2015) 8:130; Kontermann and Brinkmann. “Bispecificantibodies” Drug Discov. Today (2015) 20: 838-847). The methods ofproducing BsAbs are not limited to quadroma technology based on thesomatic fusion of two different hybridoma cell lines, chemicalconjugation, which involves chemical cross-linkers, and geneticapproaches utilizing recombinant DNA technology. Examples of bsAbsinclude those disclosed in the following patent applications, which arehereby incorporated by reference in their entirety: U.S. Pat. No.8,586,713, filed Jun. 25, 2010; U.S. Pat. Publication No. 2013/0045492,filed Jun. 5, 2012; U.S. Pat. No. 9,657,102, filed Sep. 19, 2013; U.S.Pat. Publication No. 2016/0024147, filed Jul. 24, 2015; U.S. Pat.Publication No. 2018/0112001, filed Sep. 22, 2017; U.S. Pat. PublicationNo. 2018/0104357, field Sep. 22, 2017; U.S. Pat. Publication No.2017/0174779, filed Dec. 21, 2016; U.S. Pat. Publication No.2017/0174781, filed Dec. 21, 2016; U.S. Pat. No. 10,179,819, filed Jul.29, 2016; and U.S. Pat. Publication No. 2018/0134794, filed Nov. 15,2017. Low levels of homodimer impurities can be present at several stepsduring the manufacturing of bispecific antibodies. The detection of suchhomodimer impurities can be challenging when performed using intact massanalysis due to low abundances of the homodimer impurities and theco-elution of these impurities with main species when carried out usinga regular liquid chromatographic method (as illustrated in FIG. 2).

Therapeutic bispecific antibodies (bsAbs) can simultaneously bind to twodistinct targets and hold the promise to achieve enhanced therapeuticefficacy by offering dual functionality or novel mechanisms of action(Marie Godar et al., Therapeutic bispecific antibody formats: a patentapplications review (1994-2017), 28 EXPERT OPINION ON THERAPEUTICPATENTS 251-276 (2018)). To date, more than 60 bispecific molecules havebeen developed and evaluated to treat various diseases, many of whichadopt an IgG-like architecture due to its known advantages (stability,serum half-life, etc.) in therapeutic applications (Christoph Spiess,Qianting Zhai & Paul J. Carter, Alternative molecular formats andtherapeutic applications for bispecific antibodies, 67 MOLECULARIMMUNOLOGY 95-106 (2015); M X Sliwkowski & I Mellman, Antibodytherapeutics in cancer., 341 SCIENCE 1192-1198 (2013); Paul J. Carter,Potent antibody therapeutics by design, 6 NATURE REVIEWS IMMUNOLOGY343-357 (2006)). Bispecific antibodies are frequently produced in asingle cell by co-expressing different light and heavy chains.Subsequently, assembly of the bsAb construct requires correct pairing ofcognate light and heavy chains, as well as heterodimerization of twodifferent half-molecules. Unfortunately, this process can also result inthe formation of misassembled molecular constructs, such as monospecificmolecules (e.g., homodimer species). Unlike other impurities, removal ofhomodimer species through down-stream purification can be challenging,as they often exhibit highly similar physicochemical properties to theanticipated bsAb products. To improve the fidelity of polypeptide chainpairing and therefore favoring the formation of bsAbs, variousstrategies have been developed in recent years (Shixue Chen et al.,Immunoglobulin Gamma-Like Therapeutic Bispecific Antibody Formats forTumor Therapy, 2019 JOURNAL OF IMMUNOLOGY RESEARCH 1-13 (2019)). Forexample, the use of an identical light chain for each antigen-bindingarm of the bsAb has been particularly successful to avoid the mispairingbetween light and heavy chains (A. Margaret Merchant et al., Anefficient route to human bispecific IgG, 16 NATURE BIOTECHNOLOGY 677-681(1998)). In addition, the hetero-dimerization of different heavy chainscan be greatly facilitated using a knobs-into-holes (John B.b. Ridgway,Leonard G. Presta & Paul Carter, ‘Knobs-into-holes’ engineering ofantibody CH3 domains for heavy chain heterodimerization, 9 “PROTEINENGINEERING, DESIGN AND SELECTION” 617-621 (1996)) design, wherespecific mutations were engineered into the Fc portion of the antibodyto favor heterodimer formation. Alternatively, by modulating the ProteinA binding affinity via amino acid substitutions in the Fc portion, absAb can also be effectively isolated from homodimer impurities duringthe Protein A purification step (Adam Zwolak et al., Rapid Purificationof Human Bispecific Antibodies via Selective Modulation of Protein ABinding, 7 SCIENTIFIC REPORTS 15521 (2017)). This strategy has alreadybeen successfully implemented to achieve mass production of bsAbs tosupport clinical studies (Andrew D. Tustian et al., Development ofpurification processes for fully human bispecific antibodies based uponmodification of protein A binding avidity, 8 MABS 828-838 (2016)).

Advances in novel bsAb formats through protein engineering and processdevelopment have enabled large scale production of therapeutic bsAbswith high purity. However, the presence of low-abundance homodimerimpurities in bsAb drug products can be still possible and needs to beroutinely monitored during development and in release testing. Regardedas product-related impurities, homodimer antibodies can be highlysimilar to the desired bsAb in many properties, rendering theirdetection and quantitation a unique and challenging task for currentanalytical techniques. As homodimer species usually exhibit distinctivemolecular weights compared to the corresponding bsAb, mass measurementat the intact protein level using LC-MS-based techniques has been themethod-of-choice for their characterization (R. Jeremy Woods et al.,LC-MS characterization and purity assessment of a prototype bispecificantibody, 5 MABS 711-722 (2013); Wolfgang Schaefer et al., Heavy andlight chain pairing of bivalent quadroma and knobs-into-holes antibodiesanalyzed by UHR-ESI-QTOF mass spectrometry, 8 MABS 49-55 (2015); FrankD. Macchi et al., Absolute Quantitation of Intact Recombinant AntibodyProduct Variants Using Mass Spectrometry, 87 ANALYTICAL CHEMISTRY10475-10482 (2015); Luis Schachner et al., Characterization of ChainPairing Variants of Bispecific IgG Expressed in a Single Host Cell byHigh Resolution Native and Denaturing Mass Spectrometry, 88 ANALYTICALCHEMISTRY 12122-12127 (2016); Yiyuan Yin et al., Precise quantificationof mixtures of bispecific IgG produced in single host cells by liquidchromatography-Orbitrap high-resolution mass spectrometry, 8 MABS1467-1476 (2016); Chunlei Wang et al., A systematic approach foranalysis and characterization of mispairing in bispecific antibodieswith asymmetric architecture, 10 MABS 1226-1235 (2018); Markus Habergeret al., Rapid characterization of biotherapeutic proteins bysize-exclusion chromatography coupled to native mass spectrometry, 8MABS 331-339 (2015); François Debaene et al., Time Resolved NativeIon-Mobility Mass Spectrometry to Monitor Dynamics of IgG4 Fab ArmExchange and “Bispecific” Monoclonal Antibody Formation, 85 ANALYTICALCHEMISTRY 9785-9792 (2013)). For example, the use of reversed phasechromatography (RPLC) coupled to a high-resolution accurate-mass (HRAM)mass spectrometer has been reported by several labs to quantifyhomodimer impurities in bsAb samples (Woods et al, 2013, supra;Schachner et al, 2016, supra; Yin et al, 2016, supra). In most of thesestudies, the homodimer impurities could be detected and quantifiedwithout chromatographic separation from the main bsAb species. Indeed,considering the large size (˜150 kDa) as well as the similarity inphysicochemical properties, it can often be a challenging task toachieve sufficient separation between homodimer antibodies and bsAbusing the RPLC method. As a result, RPLC-MS-based methods are frequentlylacking sensitivity in detecting homodimer species present at low levels(the lowest LLOQ reported is ˜1% (Schachner et al, 2016, supra; Yin etal, 2016, supra), largely due to ion suppression from the co-eluting andoverwhelmingly more abundant bsAb species. Moreover, withoutchromatographic separation, detection of homodimer species withmolecular weights close to bsAb can be particularly challenging, asvariant forms of the bsAb from PTMs (e.g., +128 Da for C-terminal Lysand +162 for glycation) or adduction formation, could potentiallyinterfere with the analysis. Finally, in cases where chromatographicseparation between homodimer and bsAb is achieved by the RPLC method,MS-based quantitation could still be compromised by the discrepancy inionization efficiency of antibodies eluting at different retention timeswithin different solvent compositions, which requires generating anexternal calibration curve using spiked-in standards when performingquantitation (Risto Kostiainen & Tiina J. Kauppila, Effect of eluent onthe ionization process in liquid chromatography-mass spectrometry, 1216JOURNAL OF CHROMATOGRAPHY A 685-699 (2009)). Alternatively, native massspectrometry represents another valuable technique in the analysis ofintact proteins and has been integrated into many routine analyticalworkflows for monoclonal antibody (mAb) heterogeneity assessment(Haberger et al, 2016, supra; Sara Rosati et al., Qualitative andSemiquantitative Analysis of Composite Mixtures of Antibodies by NativeMass Spectrometry, 84 ANALYTICAL CHEMISTRY 7227-7232 (2012); AnthonyEhkirch et al., Hyphenation of size exclusion chromatography to nativeion mobility mass spectrometry for the analytical characterization oftherapeutic antibodies and related products, 1086 JOURNAL OFCHROMATOGRAPHY B 176-183 (2018); Guillaume Terral, Alain Beck & SarahCianférani, Insights from native mass spectrometry and ion mobility-massspectrometry for antibody and antibody-based product characterization,1032 JOURNAL OF CHROMATOGRAPHY B 79-90 (2016); Oscar Hernandez-Alba etal., Native Mass Spectrometry, Ion Mobility, and Collision-InducedUnfolding for Conformational Characterization of IgG4 MonoclonalAntibodies, 90 ANALYTICAL CHEMISTRY 8865-8872 (2018)). Because of themore concentrated signal generated from fewer charge states, native MScan have an improved sensitivity over RPLC-MS. For example, Rosati etal. (supra) reported the use of native MS to study a binary mixture oftwo co-expressed IgG1 antibodies. However, without efficientchromatographic separation, detection and quantitation of homodimerspecies present at low levels continues to be challenging for the samereasons discussed above.

To date, there have been limited reports on analytical methods that relyon chromatographic separation for detection and quantitation ofhomodimer species in bsAb samples. For example, hydrophobic interactionchromatography (HIC) (Wang et al., 2018, supra) or ion exchangechromatography (IEX) (A. F. Labrijn et al., Efficient generation ofstable bispecific IgG1 by controlled Fab-arm exchange, 110 PROCEEDINGSOF THE NATIONAL ACADEMY OF SCIENCES 5145-5150 (2013); Michael J Grameret al., Production of stable bispecific IgG1 by controlled Fab-armexchange, 5 MABS 962-973 (2013)) have been shown to separate bsAb fromits homodimers if they exhibit sufficiently different values inhydrophobicity or isoelectric point (pI), respectively. In addition,recent advances in online IEX-native MS technologies (Yuetian Yan etal., Ultrasensitive Characterization of Charge Heterogeneity ofTherapeutic Monoclonal Antibodies Using Strong Cation ExchangeChromatography Coupled to Native Mass Spectrometry, 90 ANALYTICALCHEMISTRY 13013-13020 (2018); Florian Füssl et al., Charge VariantAnalysis of Monoclonal Antibodies Using Direct Coupled pH GradientCation Exchange Chromatography to High Resolution Native MassSpectrometry, 90 ANALYTICAL CHEMISTRY 4669-4676 (2018); Aaron O. Baileyet al., Charge variant native mass spectrometry benefits mass precisionand dynamic range of monoclonal antibody intact mass analysis, 10 MABS1214-1225 (2018)) provide an effective approach for sensitive detectionof low-level homodimer species in bsAb. However, owing to the highresolution, IEX usually generates a complicated charge profile for eachantibody based on their charge heterogeneity, which will likely overlapwith each other. Moreover, MS-based quantitation using this approach canbe complicated, as all of the separated charge variant forms from eachmolecule need to be summed up for calculation. In addition, similar tothe RPLC-based approach, the IEX method utilizes a gradient elution,which will likely compromise MS-based quantitation due to differentionization efficiency of antibodies eluting under different solventconditions (e.g., pH or salt concentrations). Recently, a SEC-basedmixed-mode chromatography (MM-SEC) method, which separates analytes byboth hydrodynamic volume and hydrophobic interactions with the columnmatrix, has been applied in studying antibody heterogeneity (Xiaoyu Yanget al., Analysis and purification of IgG4 bispecific antibodies by amixed-mode chromatography, 484 ANALYTICAL BIOCHEMISTRY 173-179 (2015);Cintyu Wong, Camille Strachan-Mills & Sudhir Burman, Facile method ofquantification for oxidized tryptophan degradants of monoclonal antibodyby mixed mode ultra performance liquid chromatography, 1270 JOURNAL OFCHROMATOGRAPHY A 153-161 (2012); Jorge Alexander Pavon et al., Analysisof monoclonal antibody oxidation by simple mixed mode chromatography,1431 JOURNAL OF CHROMATOGRAPHY A 154-165 (2016)). Coupled to UV orfluorescence detection, the MM-SEC method was successfully applied forrelative quantitation of homodimer species in a bsAb sample (Yang et al,2015, supra). However, it is clear that the utility of this method canbe limited to cases where the homodimer and bsAb species aresufficiently different in hydrophobicity so that baseline separation canbe achieved for UV- or fluorescence-based quantitation. Moreover, as theidentification of homodimer species was solely based on retention timealignment against standards, there was always a risk of overestimationof relative abundance if they co-elute with the oligomeric or truncatedforms of bsAb molecule.

The concept of mixed-mode chromatography using an SEC column originatesfrom the unwanted secondary interactions between protein analytes andthe column matrix. Ideally, SEC should separate protein analytes solelybased on their hydrodynamic volume. In practice, electrostatic,hydrophobic and hydrogen-bonding interactions could all contribute tothe retention and separation of proteins to different extents, dependingon the column matrix, buffer conditions and protein characteristics(Alexandre Goyon et al., Unraveling the mysteries of modern sizeexclusion chromatography—the way to achieve confident characterizationof therapeutic proteins, 1092 JOURNAL OF CHROMATOGRAPHY B 368-378(2018); Tsutomu Arakawa et al., The critical role of mobile phasecomposition in size exclusion chromatography of protein pharmaceuticals,99 JOURNAL OF PHARMACEUTICAL SCIENCES 1674-1692 (2010)). Utilizing thesesecondary interactions during SEC separation by properly optimizing thechromatographic conditions presents opportunities to improve separationof antibodies with similar hydrodynamic volume but different surfacecharacteristics (e.g., charge and hydrophobicity). By usingMS-compatible mobile phases, online coupling of mixed-mode SEC withnative MS detection (MM-SEC-MS) can have many advantages over UV-basedmethods, including unambiguous identification of homodimer species byaccurate mass measurements, minimal interference from co-eluting speciesand less stringent requirements on chromatographic resolution (Terral etal., 2016, supra; Goyon et al., 2017, supra).

As used herein “multispecific antibody” or “Mab” refers to an antibodywith binding specificities for at least two different antigens. Whilesuch molecules normally will only bind two antigens (i.e. bispecificantibodies, BsAbs), antibodies with additional specificities such astrispecific antibody and KIH Trispecific can also be addressed by thesystem and method disclosed herein.

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. A monoclonal antibodycan be derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, by any means available or known in the art.Monoclonal antibodies useful with the present disclosure can be preparedusing a wide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technologies, or a combinationthereof.

During many stages of production of biopharmaceuticals, impurities canbe formed. Biotechnology-derived impurities can be very difficult tocharacterize and quantify, because they often are present at very lowlevels, and because they can represent very complicated species ormixtures of species. It can be also very difficult to obtain anauthentic reference standard of the impurity peaks. However, to fullycharacterize a trace amount of an impurity protein becomes a timeconsuming, lengthy, and often very expensive process. Often the impuritycan include variants, isoforms, degradation products, product-relatedimpurities, process-related, minor post translational modifications,aggregates, or clipped fragments of the intact recombinant protein.There are an almost infinite number of possible impurities, most ofwhich might be known but not all.

As used herein, the term “target protein” can include the desiredproduct or an impurity or both.

As used herein, the term “desired product” refers to the protein whichhas the desired structure, function, or efficacy profile.

As used herein, the term “impurity” can include any undesirable proteinpresent in the biopharmaceutical product. Impurity can include processand product-related impurities. The impurity can further be of knownstructure, partially characterized, or unidentified. Process-relatedimpurities can be derived from the manufacturing process and can includethe three major categories: cell substrate-derived, cell culture-derivedand downstream derived. Cell substrate-derived impurities include, butare not limited to, proteins derived from the host organism and nucleicacid (host cell genomic, vector, or total DNA). Cell culture-derivedimpurities include, but are not limited to, inducers, antibiotics,serum, and other media components. Downstream-derived impuritiesinclude, but are not limited to, enzymes, chemical and biochemicalprocessing reagents (e.g., cyanogen bromide, guanidine, oxidizing andreducing agents), inorganic salts (e.g., heavy metals, arsenic,nonmetallic ion), solvents, carriers, ligands (e.g., monoclonalantibodies), and other leachables. Product-related impurities (e.g.,precursors, certain degradation products) can be molecular variantsarising during manufacture and/or storage that do not have propertiescomparable to those of the desired product with respect to activity,efficacy, and safety. Such variants may need considerable effort inisolation and characterization in order to identify the type ofmodification(s). Product-related impurities can include truncated forms,modified forms, and aggregates. Truncated forms are formed by hydrolyticenzymes or chemicals which catalyze the cleavage of peptide bonds.Modified forms include, but are not limited to, deamidated, isomerized,mismatched S-S linked, oxidized, or altered conjugated forms (e.g.,glycosylation, phosphorylation). Modified forms can also include anypost-translational modification form. Aggregates include dimers andhigher multiples of the desired product. (Q6B Specifications: TestProcedures and Acceptance Criteria for Biotechnological/BiologicalProducts, ICH August 1999, U.S. Dept. of Health and Humans Services).

As used herein, the general term “post-translational modifications” or“PTMs” refer to covalent modifications that polypeptides undergo, eitherduring (co-translational modification) or after (post-translationalmodification) their ribosomal synthesis. PTMs are generally introducedby specific enzymes or enzyme pathways. Many occur at the site of aspecific characteristic protein sequence (signature sequence) within theprotein backbone. Several hundred PTMs have been recorded, and thesemodifications invariably influence some aspect of a protein's structureor function (Walsh, G. “Proteins” (2014) second edition, published byWiley and Sons, Ltd., ISBN: 9780470669853). The variouspost-translational modifications include, but are not limited to,cleavage, N-terminal extensions, protein degradation, acylation of theN-terminus, biotinylation (acylation of lysine residues with a biotin),amidation of the C-terminal, glycosylation, iodination, covalentattachment of prosthetic groups, acetylation (the addition of an acetylgroup, usually at the N-terminus of the protein), alkylation (theaddition of an alkyl group (e.g., methyl, ethyl, propyl) usually atlysine or arginine residues), methylation, adenylation,ADP-ribosylation, covalent cross links within, or between, polypeptidechains, sulfonation, prenylation, Vitamin C dependent modifications(proline and lysine hydroxylations and carboxy terminal amidation),Vitamin K dependent modification wherein Vitamin K is a cofactor in thecarboxylation of glutamic acid residues resulting in the formation of aγ-carboxyglutamate (a glu residue), glutamylation (covalent linkage ofglutamic acid residues), glycylation (covalent linkage glycineresidues), glycosylation (addition of a glycosyl group to eitherasparagine, hydroxylysine, serine, or threonine, resulting in aglycoprotein), isoprenylation (addition of an isoprenoid group such asfarnesol and geranylgeraniol), lipoylation (attachment of a lipoatefunctionality), phosphopantetheinylation (addition of a4′-phosphopantetheinyl moiety from coenzyme A, as in fatty acid,polyketide, non-ribosomal peptide and leucine biosynthesis),phosphorylation (addition of a phosphate group, usually to serine,tyrosine, threonine or histidine), and sulfation (addition of a sulfategroup, usually to a tyrosine residue). The post-translationalmodifications that change the chemical nature of amino acids include,but are not limited to, citrullination (the conversion of arginine tocitrulline by deimination), and deamidation (the conversion of glutamineto glutamic acid or asparagine to aspartic acid). The post-translationalmodifications that involve structural changes include, but are notlimited to, formation of disulfide bridges (covalent linkage of twocysteine amino acids) and proteolytic cleavage (cleavage of a protein ata peptide bond). Certain post-translational modifications involve theaddition of other proteins or peptides, such as ISGylation (covalentlinkage to the ISG15 protein (Interferon-Stimulated Gene)), SUMOylation(covalent linkage to the SUMO protein (Small Ubiquitin-relatedMOdifier)) and ubiquitination (covalent linkage to the proteinubiquitin). See http://www.uniprot.org/docs/ptmlist for a more detailedcontrolled vocabulary of PTMs curated by UniProt.

Mixed Mode Size-Exclusion Chromatography

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas can be separated intocomponents as a result of differential distribution of the chemicalentities as they flow around or over a stationary liquid or solid phase.

As used herein, the term “Mixed Mode Chromatography (MMC)” or“multimodal chromatography” includes a chromatographic method in whichsolutes interact with stationary phase through more than one interactionmode or mechanism. MMC can be used as an alternative or complementarytool to traditional reversed-phased (RP), ion exchange (IEX) and normalphase chromatography (NP). Unlike RP, NP and IEX chromatography, inwhich hydrophobic interaction, hydrophilic interaction and ionicinteraction respectively are the dominant interaction modes, mixed-modechromatography can employ a combination of two or more of theseinteraction modes. Mixed mode chromatography media can provide uniqueselectivity that cannot be reproduced by single mode chromatography.Mixed mode chromatography can also provide potential cost savings andoperation flexibility compared to affinity based methods.

The phrase “size exclusion chromatography” or “SEC” or “gel filtration”includes a liquid column chromatographic technique that can sortmolecules according to their size in solution.

As used herein, the terms “SEC chromatography resin” or “SECchromatography media” are used interchangeably herein and can includeany kind of solid phase used in SEC which separates the impurity fromthe desired product (e.g., a homodimer contaminant for a bispecificantibody product). The volume of the resin, the length and diameter ofthe column to be used, as well as the dynamic capacity and flow-rate candepend on several parameters such as the volume of fluid to be treated,concentration of protein in the fluid to be subjected to the process ofthe invention, etc. Determination of these parameters for each step iswell within the average skills of the person skilled in the art.

As used herein, the term “mixed-mode-size exclusion chromatography” or“MM-SEC” can include any chromatographic method which separates proteinsthrough an additional interaction other than the separation based ontheir size. The additional or secondary interaction can exploit one ormore of the following mechanisms: anion exchange, cation exchange,hydrophobic interaction, hydrophilic interaction, charge-chargeinteraction, hydrogen bonding, pi-pi bonding, and metal affinity. Themixed-mode-size exclusion chromatography resin can refer to any kind ofsolid phase used for MM-SEC separation. Non-limiting examples are SepaxZenix SEC-300, Waters BEH 300, or Agilent Bio SEC-3.

As used herein, the term “hydrophobic functionality” refers to thehydrophobic interaction of the protein with the SEC chromatographicresin as a secondary interaction. The hydrophobic functionality can alsosignificantly impact peak shape, which can have a pronounced effect onthe resolving ability of the process. Hydrophobic interactions arestrongest at high ionic strength of the mobile phase. For selecting amobile phase to include hydrophobic functionality in a resin, variousions can be arranged in a so-called soluphobic series depending onwhether they promote hydrophobic interactions (salting-out effects) ordisrupt the structure of water (chaotropic effect) and lead to theweakening of the hydrophobic interaction (as illustrated in FIG. 3).Cations are ranked in terms of increasing salting out effect as Ba⁺⁺;Ca⁺⁺; Mg⁺⁺; Li⁺; Cs⁺; Na⁺; K⁺; Rb⁺; NH⁴⁺, while anions may be ranked interms of increasing chaotropic effect as PO⁻; SO₄ ⁻; CH₃CO₂ ⁻; Cl⁻; Br⁻;NO₃ ⁻; ClO₄ ⁻; I⁻; SCN⁻. In general, Na, K or NH₄ sulfates effectivelypromote ligand-protein interaction in HIC. Salts may be formulated thatinfluence the strength of the interaction as given by the followingrelationship:(NH₄)₂SO₄>Na₂SO₄>NaCl>NH₄Cl>NaBr>NaSCN.Mass Spectrometry

As used herein, the term “mass spectrometer” includes a device capableof detecting specific molecular species and measuring their accuratemasses. The term is meant to include any molecular detector into which apolypeptide or peptide may be eluted for detection and/orcharacterization. A mass spectrometer can include three major parts: theion source, the mass analyzer, and the detector. The role of the ionsource is to create gas phase ions. Analyte atoms, molecules, orclusters can be transferred into gas phase and ionized eitherconcurrently (as in electrospray ionization). The choice of ion sourcedepends heavily on the application.

As used herein, the term “mass analyzer” includes a device that canseparate species, that is, atoms, molecules, or clusters, according totheir mass. Non-limiting examples of mass analyzers that could beemployed for fast protein sequencing are time-of-flight (TOF),magnetic/electric sector, quadrupole mass filter (Q), quadrupole iontrap (QIT), orbitrap, Fourier transform ion cyclotron resonance (FTICR),and also the technique of accelerator mass spectrometry (AMS).

As used herein, the term “tandem mass spectrometry” includes a techniquewhere structural information on sample molecules is obtained by usingmultiple stages of mass selection and mass separation. A prerequisite isthat the sample molecules can be transferred into gas phase and ionizedintact and that they can be induced to fall apart in some predictableand controllable fashion after the first mass selection step. MultistageMS/MS, or MS^(n) can be performed by first selecting and isolating aprecursor ion (MS²), fragmenting it, isolating a primary fragment ion(MS³), fragmenting it, isolating a secondary fragment (MS⁴), and so onas long as one can obtain meaningful information or the fragment ionsignal is detectable. Tandem MS have been successfully performed with awide variety of analyzer combinations. What analyzers to combine for acertain application is determined by many different factors, such assensitivity, selectivity, and speed, but also size, cost, andavailability. The two major categories of tandem MS methods aretandem-in-space and tandem-in-time, but there are also hybrids wheretandem-in-time analyzers are coupled in space or with tandem-in-spaceanalyzers.

In some exemplary embodiments, mass spectrometry can be performed undernative conditions.

As used herein, the term “native conditions” or “native MS” or “nativeESI-MS” can include a performing mass spectrometry under conditions thatpreserve non-covalent interactions in an analyte. For detailed review onnative MS, refer to the review: Elisabetta Boeri Erba & Carlo Petosa,The emerging role of native mass spectrometry in characterizing thestructure and dynamics of macromolecular complexes, 24 PROTEIN SCIENCE1176-1192 (2015). Some of the distinctions between native ESI andregular ESI are illustrated in table 1 (Hao Zhang et al., Native massspectrometry of photosynthetic pigment-protein complexes, 587 FEBSLetters 1012-1020 (2013)).

TABLE 1 Native ESI Regular ESI Sample Aqueous solution Partial organicsolution Solution water, ammonium acetate water, formic acid,acetonitrile/Methanol (pH 1-2) Spray 10-50 nL/min 10-50 nL/min ConditionSpray voltage 0.8-1.5 kV Spray voltage 0.8-1.5 kV Temperatures 20-30° C.Temperatures 20-30° C. Salt Treatment Offline Desalt Online/OfflineDesalt with RP-HPLC Protein 1-10 μM (complex) <1 μM (subunit)Concentration Output Molecular weight of protein Molecular weight of asingle Information complex and subunit subunit Non-covalent interactionsStoichiometry StructureExamplary Embodiments

Embodiments disclosed herein provide compositions, methods, and systemsfor the rapid characterization of proteins in a sample.

As used herein, the terms “include,” “includes,” and “including,” aremeant to be non-limiting and are understood to mean “comprise,”“comprises,” and “comprising,” respectively.

The disclosure provides methods for detecting or quantifying an impurityin a sample comprising contacting the sample to a chromatographic systemhaving a mixed-mode chromatography resin; washing the mixed-mode sizeexclusion chromatography resin using a mobile phase to provide an eluentincluding the impurity; and detecting the impurity in the eluent using amass spectrometer.

The disclosure provides methods for detecting or quantifying a targetprotein in a sample comprising contacting the sample to achromatographic system having a mixed-mode chromatography resin, washingthe mixed-mode chromatography resin using a mobile phase to provide aneluent including the target protein, and detecting or quantifying thetarget protein in the eluent using a mass spectrometer.

In some specific exemplary embodiments, the chromatographic system cancomprise a size exclusion chromatography resin with an additionalinteraction.

In some specific exemplary embodiments, the chromatographic system cancomprise a size exclusion chromatography resin with a hydrophobicinteraction functionality.

In some specific exemplary embodiments, the chromatographic system cancomprise a size exclusion chromatography resin with a charge-chargeinteraction functionality.

In some exemplary embodiments, the method for detecting or quantifyingan impurity in a sample can include an impurity which can include atleast one undesirable protein. The impurity(s) can be of knownstructure, or be partially characterized, or be unidentified.

In some exemplary embodiments, the impurity can be a product-relatedimpurity. The product related impurity can be molecular variants,precursors, degradation products, fragmented protein, digested product,aggregates, post-translational modification form, or combinationsthereof.

In some specific exemplary embodiments, the impurity can be aprocess-related impurity. The process-related impurity can includeimpurities derived from the manufacturing process, i.e., nucleic acidsand host cell proteins, antibiotics, serum, other media components,enzymes, chemical and biochemical processing reagents, inorganic salts,solvents, carriers, ligands, and other leachables used in themanufacturing process.

In some exemplary embodiments, the impurity can be a protein with a pIin the range of about 4.5 to about 9.0. In one aspect, the impurity canbe a protein with a pI of about 4.5, about 5.0, about 5.5, about 5.6,about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 about 6.2, about6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9,about 7.0, about 7.1 about 7.2, about 7.3, about 7.4, about 7.5, about7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1 about 8.2,about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about8.9, or about 9.0.

In some exemplary embodiments, the impurity can be a homodimer species.In one aspect, the impurity can be a homodimer species, which can beformed during the production of a bispecific antibody. In anotheraspect, the number of impurities in the sample can be at least two.

In some exemplary embodiments, amount of the sample loaded on thechromatographic system can range from about 10 μg to about 100 μg. Inone exemplary embodiment, the amount of the sample loaded on thechromatographic system can be about 10 μg, about 12.5 μg, about 15 μg,about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg,about 75 μg, about 80 μg, about 85 μg, about 90 μg, about 95 μg, orabout 100 μg.

In some exemplary embodiments, the mobile phase used to elute theimpurity can be a mobile phase that can be compatible with a massspectrometer.

In some specific exemplary embodiments, the mobile phase can be ammoniumacetate, ammonium bicarbonate, or ammonium formate, or combinationsthereof. In one aspect, the total concentration of the mobile phase canrange up to about 600 mM. In a specific aspect, the total concentrationof the mobile phase can be about 5 mM, about 6 mM, 7 mM, about 8 mM, 9mM, about 10 mM, 12.5 mM, about 15 mM, 17.5 mM, about 20 mM, 25 mM,about 30 mM, 35 mM, about 40 mM, 45 mM, about 50 mM, 55 mM, about 60 mM,65 mM, about 70 mM, 75 mM, about 80 mM, 75 mM, about 95 mM, 100 mM,about 1100 mM, 120 mM, about 130 mM, 140 mM, about 150 mM, 160 mM, about170 mM, 180 mM, about 190 mM, 200 mM, about 225 mM, 250 mM, about 275mM, 300 mM, about 325 mM, 350 mM, about 375 mM, 400 mM, about 425 mM,450 mM, about 475 mM, 500 mM, about 525 mM, 550 mM, about 575 mM, orabout 600 mM.

In some exemplary embodiments, the mobile phase can have a flow rate ofabout 0.1 ml/min to about 0.4 ml/min. In one exemplary embodiment, theflow rate of the mobile phase can be about 0.1 ml/min, about 0.15ml/min, about 0.20 ml/min, about 0.25 ml/min, about 0.30 ml/min, about0.35 ml/min, or about 0.4 ml/min.

In some exemplary embodiments, the method for detecting or quantifyingan impurity can comprise detecting or quantifying the impurity in eluentusing a mass spectrometer. In one aspect, the mass spectrometer can be atandem mass spectrometer. In another aspect, the mass spectrometer cancomprise a nano-spray.

In some exemplary embodiments, the eluent can comprise a target proteinin addition to the impurity. In one aspect, the target protein caninclude an antibody, bispecific antibody, antibody fragment, or amultispecific antibody. In a specific aspect, the target protein can bea monoclonal antibody. In a specific aspect, the target protein can be atherapeutic antibody. In a specific aspect, the target protein can be animmunoglobulin protein. In another specific aspect, immunoglobulinprotein can be IgG1. In yet another specific aspect, immunoglobulinprotein can be IgG4. In one aspect, the target protein can be abispecific antibody. In a specific aspect, the bispecific antibody canbe Anti-CD20/CD3 monoclonal antibody. In one aspect, the target proteincan be an antibody generated using mouse fibroblast cell line MG87. Inone aspect, the target protein can be an antibody fragment formed ondigestion of the antibody.

In one aspect, the target protein can be a post-translationally modifiedprotein. In a specific aspect, the post-translationally modified proteincan be a formed by cleavage, N-terminal extensions, protein degradation,acylation of the N-terminus, biotinylation, amidation of the C-terminal,oxidation, glycosylation, iodination, covalent attachment of prostheticgroups, acetylation, alkylation, methylation, adenylation,ADP-ribosylation, covalent cross links within, or between, polypeptidechains, sulfonation, prenylation, Vitamin C dependent modifications,Vitamin K dependent modification, glutamylation, glycylation,glycosylation, deglycosylation, isoprenylation, lipoylation,phosphopantetheinylation, phosphorylation, sulfation, citrullination,deamidation, formation of disulfide bridges, proteolytic cleavage,ISGylation, SUMOylation or ubiquitination (covalent linkage to theprotein ubiquitin).

In another aspect, the target protein can be a degradation product of aprotein.

In yet another aspect, the target protein can be an impurity found in abiopharmaceutical product. In a specific aspect, the target protein canbe an impurity found during the manufacture of the biopharmaceuticalproduct.

In one aspect, the target protein can be a protein with a pI in therange of about 4.5 to about 9.0.

In one aspect, the target protein can be a product-related impurity. Theproduct related impurity can be molecular variants, precursors,degradation products, fragmented protein, digested product, aggregates,post-translational modification form, or combinations thereof.

In one aspect, the target protein can be a process-related impurity. Theprocess-related impurity can include impurities derived from themanufacturing process, i.e., nucleic acids and host cell proteins,antibiotics, serum, other media components, enzymes, chemical andbiochemical processing reagents, inorganic salts, solvents, carriers,ligands, and other leachables used in the manufacturing process.

In one aspect, the number of impurities in the sample can be at leasttwo.

In one aspect, the post-transnationally modified protein can be formedon oxidation of a protein.

In another aspect, the target protein can include a degradation product.

In another aspect, the degradation product can include apost-translation modification of a therapeutic protein.

In some exemplary embodiments, washing the mixed-mode chromatographyresin using a mobile phase requires less than about 30 minutes. In oneaspect, the time required for washing the mixed-mode chromatographyresin using a mobile phase can be about 10 minutes, about 11 minutes,about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes,about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes,about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes,about 24 minutes, about 25 minutes, about 26 minutes, about 26 minutes,about 27 minutes, about 28 minutes, about 29 minutes, or about 30minutes.

In some exemplary embodiments, the chromatographic system can be usedfor at least about 3 sample runs without cleaning. In one aspect, thechromatographic system can be used for at least about 3 sample runs, atleast about 4 sample runs, at least about 5 sample runs, at least about6 sample runs, at least about 7 sample runs, or at least about 8 sampleruns, without cleaning.

It is understood that the methods are not limited to any of theaforesaid protein, impurity, column and that the methods for detectingor quantifying may be conducted by any suitable means.

In some exemplary embodiments, the disclosure provides a mixed-modechromatographic system comprising a chromatographic column 110 capableof being washed using a mobile phase to provide an eluent including atarget protein and a mass spectrometer 120 coupled to thechromatographic column (as illustrated in FIG. 4). In one aspect, thechromatographic column 110 can be capable of being contacted with asample using a sample loading device 100.

In some exemplary embodiments, the amount of the sample that can beloaded on the chromatographic column 110 can range from about 10 μg toabout 100 μg. In one aspect, the amount of the sample that can be loadedon the chromatographic column 110 can be about 10 μg, about 12.5 μg,about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg,about 70 μg, about 75 μg, about 80 μg, about 85 μg, about 90 μg, about95 μg, or about 100 μg.

In some exemplary embodiments, the chromatographic column 110 can becapable of being washed with a mobile phase. In one aspect, the mobilephase can be ammonium acetate, ammonium bicarbonate, or ammoniumformate, or combinations thereof. In one aspect, the total concentrationof the mobile phase that can be used with the chromatographic column 110can range up to about 600 mM. In a specific aspect, the totalconcentration of the mobile phase that can be used with thechromatographic column 110 can be about 5 mM, about 6 mM, 7 mM, about 8mM, 9 mM, about 10 mM, 12.5 mM, about 15 mM, 17.5 mM, about 20 mM, 25mM, about 30 mM, 35 mM, about 40 mM, 45 mM, about 50 mM, 55 mM, about 60mM, 65 mM, about 70 mM, 75 mM, about 80 mM, 75 mM, about 95 mM, 100 mM,about 1100 mM, 120 mM, about 130 mM, 140 mM, about 150 mM, 160 mM, about170 mM, 180 mM, about 190 mM, 200 mM, about 225 mM, 250 mM, about 275mM, 300 mM, about 325 mM, 350 mM, about 375 mM, 400 mM, about 425 mM,450 mM, about 475 mM, 500 mM, about 525 mM, 550 mM, about 575 mM, orabout 600 mM. In another aspect, the mobile phase that can be used withthe chromatographic column 110 can have a flow rate of 0.1 ml/min to 0.4ml/min. In a specific aspect, the flow rate of the mobile phase that canbe used with the chromatographic column 110 can be about 0.1 ml/min,about 0.15 ml/min, about 0.20 ml/min, about 0.25 ml/min, about 0.30ml/min, about 0.35 ml/min, or about 0.4 ml/min. In another aspect, themobile phase that can be used with the chromatographic column 110 can beused to elute the impurity.

In some exemplary embodiments, the chromatographic column 110 can becapable of being coupled with a mass spectrometer 120. In one aspect,the mass spectrometer 120 can comprise a nano-spray.

In some exemplary embodiments, the mass spectrometer 120 can be a tandemmass spectrometer.

In some exemplary embodiments, the mass spectrometer 120 can be a nativemass spectrometer.

In some exemplary embodiments, the mixed-mode chromatographic system canbe capable of detection 140 and/or quantification 130 of a targetprotein (See FIG. 4). In one aspect, the mixed-mode chromatographicsystem can be used for detection 140 and/or quantification 130 of onetarget protein.

In some exemplary embodiments, the mixed-mode chromatographic system canbe capable of detection 140 and/or quantification 130 of a monoclonalantibody.

In some exemplary embodiments, the mixed-mode chromatographic system canbe capable of detection 140 and/or quantification 130 of a therapeuticantibody.

In some exemplary embodiments, the mixed-mode chromatographic system canbe capable of detection 140 and/or quantification 130 of animmunoglobulin protein. In one aspect, the mixed-mode chromatographicsystem can be capable of detection 140 and/or quantification 130 of anIgG1 protein. In another aspect, the mixed-mode chromatographic systemcan be capable of detection 140 and/or quantification 130 of an IgG4protein. In another aspect, the mixed-mode chromatographic system cancapable of detection 140 and/or quantification 130 of a bispecificantibody. In yet another aspect, the mixed-mode chromatographic systemcan capable of detection 140 and/or quantification 130 of anAnti-CD20/CD3 monoclonal antibody.

In some exemplary embodiments, the mixed-mode chromatographic system canbe capable of detection 140 and/or quantification 130 of an antibodyfragment formed on digestion of the antibody. In one aspect, themixed-mode chromatographic system can be capable of detection 140 and/orquantification 130 of a target protein, which can be apost-translationally modified protein. In another aspect, the mixed-modechromatographic system can be capable of detection 140 and/orquantification 130 of a target protein, which can be a degradationproduct of a protein. In yet another aspect, the mixed-modechromatographic system can be capable of detection 140 and/orquantification 130 of a target protein which can be an impurity found ina biopharmaceutical product. In another aspect, the mixed-modechromatographic system can be capable of detection 140 and/orquantification 130 of a target protein which can be an impurity foundduring the manufacture of the biopharmaceutical product.

In some exemplary embodiments, the mixed-mode chromatographic system canbe capable of detection 140 and/or quantification 130 of a targetprotein which can be a protein with a pI in the range of about 4.5 toabout 9.0.

In some exemplary embodiments, the mixed-mode chromatographic system canbe capable of detection 140 and/or quantification 130 of a targetprotein which can be a product-related impurity. The product relatedimpurity can be molecular variants, precursors, degradation products,fragmented protein, digested product, aggregates, post-translationalmodification form, or combinations thereof.

In some specific exemplary embodiments, the mixed-mode chromatographicsystem can be capable of detection 140 and/or quantification 130 of atarget protein which can be a process-related impurity. Theprocess-related impurity can include impurities derived from themanufacturing process, i.e., nucleic acids and host cell proteins,antibiotics, serum, other media components, enzymes, chemical andbiochemical processing reagents, inorganic salts, solvents, carriers,ligands, and other leachables used in the manufacturing process. In oneaspect, the number of impurities in the sample can be at least two.

In some exemplary embodiments, the chromatographic column 110 capable ofbeing used for at least about 3 sample runs without cleaning. In oneaspect, the chromatographic column 110 can be used for at least about 3sample runs, at least about 4 sample runs, at least about 5 sample runs,at least about 6 sample runs, at least about 7 sample runs, or at leastabout 8 sample runs, without cleaning.

It is understood that the system is not limited to any of the aforesaidprotein, impurity, mobile phase, or chromatographic column.

The consecutive labeling of method steps as provided herein with numbersand/or letters is not meant to limit the method or any embodimentsthereof to the particular indicated order.

Various publications, including patents, patent applications, publishedpatent applications, accession numbers, technical articles and scholarlyarticles are cited throughout the specification. Each of these citedreferences is incorporated by reference, in its entirety and for allpurposes, herein.

The disclosure will be more fully understood by reference to thefollowing Examples, which are provided to describe the disclosure ingreater detail. They are intended to illustrate and should not beconstrued as limiting the scope of the disclosure.

EXAMPLES

Materials. Deionized water was provided by a Milli-Q integral waterpurification system installed with MilliPak Express 20 filter(MilliporeSigma, Burlington, Mass.). Ammonium acetate (LC/MS grade),acetic acid and ammonium bicarbonate (LC/MS grade) were purchased fromSigma-Aldrich (St. Louis, Mo.). Peptide N-glycosidase F (PNGase F) waspurchased from New England Biolabs Inc (Ipswich, Mass.). Invitrogen™UltraPure™ 1 M Tris-HCl buffer, pH 7.5 was obtained from Thermo FisherScientific (Waltham, Mass.). All monoclonal antibodies and bispecifics,including IgG1 and IgG4 subclasses were produced at Regeneron.

Sample Preparation. To reduce the mass heterogeneity introduced by thepresence of two N-linked glycans in the Fc portion, each antibody orantibody mixture sample was treated with PNGase F (1 IUB milliunit per10 μg of protein) at 45° C. in 100 mM Tris-HCl (pH 7.5) for 1 hour. Toprepare the spike-in standards of bsAb mixtures, the bsAb drug substancewas first further purified using analytical strong cation exchangechromatography (SCX) to remove any residual homodimer impurities fromthe large-scale manufacturing process. The detailed conditions for SCXfractionation are shown below. After fractionation, both the purifiedBsAb and the corresponding homodimer standards were buffer exchangedinto 50 mM of Tris-HCl buffer (pH 7.5) and each adjusted to 6 μg/μLbased on concentrations determined by Nanodrop (Thermo FisherScientific, Bremen, Germany). Subsequently, the bsAb and the twocorresponding homodimers were mixed at a ratio of 1:1:1. Finally,sequential dilutions were performed using a 2 μg/μL bsAb solution toprepare a series of spike-in standards with the homodimer levels rangingfrom 0.1% to 10%.

Purification of bsAb from the bsAb drug substance by SCX. For furtherpurification of the bsAb from each bsAb sample, analytical strong cationexchange chromatography (SCX) was performed on a Waters I-Class UPLCsystem equipped with photodiode array (PDA) detector (Waters, Milford,Mass., US). Prior to sample injection, the column compartmenttemperature was set at 45° C. and a YMC-BioPro SP-F strong cationexchange column (100 mm×4.6 mm, 5 μm) (YMC Co., LTD., Kyoto, Japan) waspreconditioned with mobile phase A (20 mM ammonium acetate, pH adjustedto 5.6 with 20 mM acetic acid) at a flow rate of 0.4 mL/min. Upon theinjection of an aliquot (200 μg) of the protein samples, the gradient isheld at 100% mobile phase A for 2 minutes followed by a linear increaseto 100% mobile phase B (140 mM ammonium acetate, 10 mM ammoniumbicarbonate, pH 7.4) in 16 minutes. The gradient was held at 100% mobilephase B for 4 minutes and then returned to 100% mobile phase A torecondition the column for 7 minutes before the next injection. Thefractionated BsAb was then buffer exchanged into the same buffer as themispaired homodimer samples before mixing to prepare the spike-instandards.

MM-SEC-MS Method. Mixed-mode size exclusion chromatography was performedon a Waters I-Class UPLC system equipped with photodiode array (PDA)detector (Waters, Milford, Mass., US). A Thermo Exactive Plus EMR massspectrometer equipped with a Nanospray Flex™ Ion Source (Thermo FisherScientific, Bremen, Germany) was used for mass measurement. Prior tosample injection, the column (Waters BEH200 SEC 4.6×300 mm, 200 Å, 1.7μm or Sepax Zenix SEC-300 4.6×300 mm, 300 Å, 3 μm) was pre-equilibratedat a flow rate of 0.2 mL/min using ammonium acetate- and ammoniumbicarbonate-based mobile phase of varying concentrations (30 mM to 450mM). This was achieved by running at a fixed percentage of mobile phaseB using a dual solvent system (mobile phase A: water; mobile phase B:420 mM ammonium acetate and 30 mM ammonium bicarbonate). Upon injectionof an antibody sample (2-10 μg), an isocratic elution method was run for24 minutes. To enable simultaneous UV and MS detection, a post-columnsplitter (˜200:1 ratio) was applied after the SEC separation to reducethe flow to ˜1 μL/min for nano-ESI-MS analysis, while diverting theremaining high flow to the PDA detector for UV monitoring at 280 nm. Adisposable PicoTip Emitter (non-coated, tip: 10±1 μm) (New Objective,Inc., Woburn, Mass., US) was used to achieve nano-ESI. For the massspectrometric analysis, the resolution was set at 17,500, the capillaryspray voltage was set at 1.5 kV, the in-source fragmentation energy wasset at 100, the collision energy was set at 10, the capillarytemperature was set at 350° C., the S-lens RF level was set at 200 andthe HCD trapping gas pressure was set at 3. Mass spectra were acquiredwith an m/z range window between 2000 and 15000.

Example 1. Secondary Interactions During SEC Using MS-Compatible Buffer

A protein surface is highly heterogeneous and consists of many differentfunctional groups that can contribute to hydrogen bonding (hydroxyl,amine and amide groups), electrostatic (charged groups), and hydrophobic(hydrophobic groups) interactions with the silica- orpolysaccharide-based SEC column matrix. These interactions, in general,have different binding strength and are highly dependent on the pH,temperature and mobile phase composition used in the SEC application.For example, when performed at near neutral pH, the silanol groups froma silica-based SEC column can be negatively charged, and thereforepromote an electrostatic interaction with basic proteins. To suppresssuch an interaction, mobile phases with moderate ionic strength (Goyonet al, 2018, supra) or low pH (less than 5) (Pavon et al, 2016, supra)were frequently required in the past.

More recently, via silica surface derivatization (such as short alkylchains or linkage of functional groups), the residual silanol groupsfrom a modern SEC column can be effectively shielded, and therefore,dramatically reduce the presence of electrostatic interactions. However,those newly introduced chemical groups might also result in otherenhanced secondary interactions (e.g., hydrophobic interaction) with theprotein analyte, as reported in recent studies (Yang et al, 2015, supra;Yap He et al., On-line coupling of size exclusion chromatography withmixed-mode liquid chromatography for comprehensive profiling ofbiopharmaceutical drug product, 1262 JOURNAL OF CHROMATOGRAPHY A 122-129(2012)). As summarized by Arakawa et al., electrostatic interactionsdominate at low salt concentrations, whereas hydrophobic interactionsare favored with high ionic strength mobile phases, particularly whenhigher-ranking salts in the Hofmeister series (See FIG. 3) are used.Although the choice of salts compatible with online SEC-MS applicationis generally limited (e.g., ammonium formate, ammonium acetate andammonium bicarbonate), different types of secondary interactions betweenthe protein analytes and the column matrix might still be modulated byvarying salt concentrations and explored for protein separationpurposes.

To assess the mixed-mode interactions associated with saltconcentration, a mixture of eight antibodies (both IgG1 and IgG4subclasses) with different surface characteristics were analyzed on aWaters BEH200 SEC column using ammonium acetate- and ammoniumbicarbonate-based mobile phases of varying concentrations from 30 mM to300 mM, a range that is feasible for subsequent native MS analysis. Themolar ratio between ammonium acetate and ammonium bicarbonate was keptconstant at 14:1 to achieve a pH value of approximately 7.4. Thechromatographic behavior of the eight mAbs were illustrated by plottingtheir SEC retention times, as determined by extracted ion chromatograms(XICs), against the mobile phase concentration (FIG. 5). Overall, theeight mAbs were most well separated when SEC was performed using amobile phase salt concentration of 30 mM. As the salt concentrationincreased, the elution profiles of the eight mAbs became more convergedand less resolved (insets in FIG. 5). These shifts in retention timescould be explained by the changes in secondary interactions between themAb molecule and the column matrix. On one hand, as the residual silanolgroups from the column matrix became negatively charged at pH 7.4, theycould interact with the positively charged protein surface viaelectrostatic interaction. This interaction gets enhanced when lowersalt concentration is used. On the other hand, hydrophobic interactionbetween the mAb molecule and the column matrix could be promoted whenhigher salt concentrations are used. Interestingly, based on their pIvalues, these eight mAbs can be categorized into three different groups,where similar relationships between the retention time and saltconcentration were observed. The first group includes the three acidicmAb molecules, mAb5 (pI=6.3), mAb6 (pI 6.4) and mAb1 (pI=6.7), which areexpected to bear fewer positive charges on the protein surface at pH7.4, and therefore exhibited the least electrostatic interactions withthe column matrix. As shown in FIG. 5, these three molecules all showedan increasing trend of retention time as the salt concentrationincreased, indicating that as weak electrostatic interactions areeliminated at higher ionic strength, the hydrophobic interaction plays adominant role during the SEC separation. On the contrary, the threebasic mAb molecules, mAb3 (pI=8.0), mAb4 (pI=8.3) and mAb8 (pI=7.6),which are expected to bear more positive charges on the protein surfaceat pH 7.4, all showed a decreasing trend in retention time as the saltconcentration increased. This inverse correlation between the saltconcentration and retention time was mainly attributed to the dominatingelectrostatic interaction, which was promoted at low salt concentrationand suppressed at high salt concentration. Lastly, unlike either theacidic or basic mAbs, mAb2 (pI=7.3) and mAb7 (pI=6.9) represent a“neutral” group, which likely bear a moderate amount of positive chargeson the protein surface, and therefore exhibited a medium level ofelectrostatic interactions with the column matrix. This group ofmolecules maintained a relatively unchanged retention time at varyingsalt concentrations (30 mM to 300 mM). In this case, as the saltconcentration increased, the increase in hydrophobic interaction waslikely close to and counteracting the decrease in electrostaticinteraction, leading to little shift in retention time. These resultsindicated that by properly modulating the salt concentrations, it mightbe possible to separate or partially separate different antibodies(e.g., bsAb vs. homodimers) using mixed-mode interactions on a SECcolumn for subsequent MS analysis.

Although it appears that greater chromatographic resolution was achievedat low salt concentrations on a Waters BEH column, caution needs to betaken for basic mAbs, as severe peak tailing may occur under thoseconditions and could significantly impact the protein recovery(Alexandre Goyon et al., Characterization of 30 therapeutic antibodiesand related products by size exclusion chromatography: Feasibilityassessment for future mass spectrometry hyphenation, 1065-1066 JOURNALOF CHROMATOGRAPHY B 35-43 (2017)).

Example 2. Detection of O-Glycan Variant of a Bispecific AntibodyBispecific Ab Using MM-SEC-MS

2.1 Sample Preparation of Bispecific Antibody

The anti-CD20× anti-CD3 Bispecific Antibody (BsAb1) is ahinge-stabilized CD20×CD3 bispecific full-length antibody (Ab) based onan IgG4 isotype modified to reduce Fc binding. It is designed to bind Tcells (via CD3) and CD20-expressing cells. The Bispecific Antibody wasproduced by following the methodology as described by Smith et al. (Sci.Rep. (2015) 5:17943).

2.2 MM-SEC-MS

The analysis using MM-SEC-MS was performed isocratically using a ZenixSEC-300 MK column (7.8×300 nm, 3 μm) on the system as described above.Elution was monitored by UV at 280 nm.

Two sets of experiments were carried out. In the first experiment, themobile phase comprised 140 mM ammonium acetate and 10 mM ammoniumbicarbonate and in the second experiment, the mobile phase comprised 420mM ammonium acetate and 30 mM ammonium bicarbonate. The elution wascarried out at a flow rate of 0.4 mL/min. The equilibration wasperformed using the mobile phase.

For analytical runs, the injection loads consisted of 100 μg of thetotal protein. The elution was carried out using an isocratic gradientconsisting of ammonium acetate (buffer A) and ammonium bicarbonate(buffer B). The mass spectrometry data was analyzed by using Intactsoftware from Protein Metrics.

The two runs with mobile phases of differing concentration revealed thathigher salt concentration can enhance the hydrophobic interaction duringSEC separation as observed from elution time of the bispecific antibodyin different mobile phases. This effect led to an increased separationbetween the bispecific antibody and its O-glycan variant (See FIG. 6).

Example 3. Detection of Homodimer Species Using Zenix SEC-300, 3 μm, 300Å, 7.8×300 mm

3.1 Sample Preparation of Bispecific Antibody and Homodimers MixtureStandards

Two homodimer impurities are generated during the production of thebispecific antibody (BsAb1) (Fc*/Fc): homodimer 1 (Fc*-Fc*) andhomodimer 2 (Fc/Fc) (See FIG. 2).

3.2 MM-SEC-MS

The acquisition using MM-SEC-MS was performed isocratically using aZenix SEC-300 MK column (7.8×300 nm, 3 μm) on the system as describedabove. Elution was monitored by UV at 280 nm.

Two set of experiments were carried out. In the first experiment, themobile phase comprised 140 mM ammonium acetate and 10 mM ammoniumbicarbonate and in the second experiment, the mobile phase comprised 420mM ammonium acetate and 30 mM ammonium bicarbonate. The elution wascarried out at a flow rate of 0.4 mL/min.

For analytical runs, the injection loads consisted of 50 μg of the totalprotein. The elution was carried out using an isocratic gradientconsisting of ammonium acetate (buffer A) and ammonium bicarbonate(buffer B). Similar to results obtained form 2.2, the two runs withmobile phases of differing concentration revealed that higher saltconcentration can enhance the hydrophobic interaction during SECseparation as observed from the elution times of the bispecific antibodyand the homodimers in the different mobile phases. The mobile phase withtotal salt concentration of 450 mM performed an improved separation ofthe homodimer 1 and homodimer 2 from the bispecific antibody (See FIG.7).

Example 4. Comparison of the Detection of Homodimer Impurities in theBispecific Antibody Using MM-SEC-MS, SEC-MS and RP LC-MS

4.1 Sample Preparation of Bispecific Antibody and Homodimers MixtureStandards

The sample was prepared using the methodology illustrated in example 3.

4.2 RP LC-MS

The sample was diluted to 0.5 mg/mL. This solution was injected at 0.5μs for LC-MS analysis. The LC-MS experiment was performed onThermoFisher Fusion Lumos Tribrid mass spectrometer. The WatersBioResolve™ mAb Polyphenyl, 450 Å, 2.7 μm 2.1×50 mm Column (P.N.186008944) was used for reverse phase separation. The sample temperaturewas set at 5° C. and column temperature was set at 80° C. The mobilephase A was 0.1% FA in water, mobile phase B was 0.1% FA inacetonitrile. The mass spectrometry experiment was performed in positivemode. The MS ion source conditions were set as the following: sprayvoltage at 3.8 kV, ion transfer tube temperature at 325° C., vaporizertemperature at 250° C., sheath gas at 40 (Arb), Aux gas at 10 (Arb),sweep gas at 2 (Arb), RF Lens (%) at 60 and source fragmentation energyat 40 V. MS data were acquired by orbitrap in high mass range mode withm/z range at 1500-4000. Resolution was set to 15,000 at m/z 200 with 10microscans, AGC target was 10⁵, maximum injection time was 50 ms. Themass spectrometry data was analyzed by using Xaclibur software.

4.3 SEC-MS

Size exclusion chromatography (SEC) was performed on the ACQUITY UPLCProtein BEH SEC Column (200 Å, 1.7 μm, 4.6 mm×300 mm) using mobile phasecomprised 140 mM ammonium acetate and 10 mM ammonium bicarbonate. TheSEC experiments were performed on a Waters Acquity UPLC I-class systemat room temperature, with wavelength detection at 280 nm, a 0.2 mL/minflow rate, and 50 μg protein injection load.

4.4 MM-SEC-MS

The analytical run on the MM-SEC-MS system was carried out isocraticallyusing a mobile phase containing 420 mM ammonium acetate and 30 mMammonium bicarbonate using a methodology illustrated in Example 3.2.

4.5 Results

Comparison of the total ion chromatogram (TIC) and the native MS spectrafor RP LC-MS on Lumos (See FIG. 8), SEC-MS on EMR, and MM-SEC-MS on EMRshows the significant separation and detection of homodimers from thebispecific antibody. The raw mass spectrogram from RP LC-MS was unableto differentiate between the homodimers and the bispecific antibody. Theraw mass spectrum from SEC-MS was able to separate and detect homodimer1 and the bispecific antibody, but the separation was not sufficient toseparate and detect homodimer 2 and the bispecific antibody. Only theraw mass spectra from MM-SEC-MS showed sufficient separation anddetection of homodimer 1, homodimer 2 and the bispecific antibody. Thiscomparison provides a proof of concept of superiority of MM-SEC-MS overSEC-MS and RP LC-MS for detection of impurities in biopharmaceuticalproducts.

Example 5. Consecutive Runs of MM-SEC-MS Detection Using Zenix SEC-300,3 μm, 300 Å, 7.8×300 mm

To evaluate the data quality of detection in consecutive runs, threeanalytical runs of the sample containing Bispecific Ab, homodimer 1, andhomodimer 2 (prepared as illustrated in 4.1) was carried out using theMM-SEC-MS system. The analytical runs were performed using themethodology illustrated in 4.2 and mobile phase comprising 280 mMammonium acetate and 20 mM ammonium bicarbonate.

The raw mass spectra and the extracted ion chromatogram (XIC) for thethree runs showed a decrease in data quality and signal to noise ratio(See FIG. 9). This effect could be because of the use of a large column(7.8×300 mm) which requires a large amount of protein sample (˜50 μg) toensure MS intensity, which can lead to protein precipitation at the highsalt concentration and therefore requires more frequent cleaning of theflow pathway (max 3 samples run before cleaning).

Further, the large column and relative low flow rate the nanosplittercan handle (max˜0.4 mL/min) led to broad peak width (˜1.5 min), whichaffects the MS intensity and resolution in some cases. The late elutiontime also slowed down overall analysis time (30 min each sample).

Example 6. Detection of Homodimer Species Using Zenix SEC-300, 3 μm, 300Å, 4.6×300 mm

6.1 Sample Preparation of Bispecific Antibody and Homodimers MixtureStandards

The Bispecific Antibody and homodimers mixture standards can be preparedby methods illustrated in 3.1

6.2 MM-SEC-MS

The acquisition using MM-SEC-MS was performed isocratically using aZenix SEC-300 MK column (4.6×300 nm, 3 μm) on the system as describedabove. Elution was monitored by UV at 280 nm.

Four set of experiments were carried out. In the first experiment, themobile phase comprised 140 mM ammonium acetate and 10 mM ammoniumbicarbonate, in the second experiment, the mobile phase comprised 280 mMammonium acetate and 20 mM ammonium bicarbonate, in the thirdexperiment, the mobile phase comprised 420 mM ammonium acetate and 30 mMammonium bicarbonate and in the second experiment, the mobile phasecomprised 560 mM ammonium acetate and 40 mM ammonium bicarbonate. Theelution was carried out at a flow rate of 0.3 mL/min.

For analytical runs, the injection loads consisted of 10 μg of theprotein. The elution was carried out using an isocratic gradientconsisting of ammonium acetate (buffer A) and ammonium bicarbonate(buffer B). The use of concentrations greater than 150 mM total saltconcentration shows an improved separation and detection of thehomodimers and bispecific antibody (See FIG. 10). The total time foranalysis on using the smaller column (4.6×300 nm) decreased to about 18minutes and the peak width decrease to less than 1 minute, compared tothe total time for analysis and peak width on using the larger column(7.8×300 nm). Representations of the differences are shown in FIG. 11.

Example 7. MM-SEC-MS Analysis of Deglycoslyated Mixture of BispecificAntibody, Homodimer 1, and Homodimer 2 on Zenix-SEC Column

7.1 Preparation of Deglycoslyated Mixture of Bispecific Antibody,Homodimer 1, and Homodimer 2

Each protein was treated with peptide N-glycosidase F (PNGase F; 1 IUBmilliunit per 10 of protein) at 45° C. for 1 hour to completely removethe glycan chains from each heavy chain constant region.

7.2 MM-SEC-MS

The acquisition using MM-SEC-MS was performed isocratically using aZenix SEC-300 MK column (4.6×300 nm, 3 μm) on the system as describedabove. Elution was monitored by UV at 280 nm.

Six set of experiments were carried out. In the first experiment, themobile phase comprised 9.3 mM ammonium acetate and 0.7 mM ammoniumbicarbonate, in the second experiment, the mobile phase comprised 46.7mM ammonium acetate and 3.3 mM ammonium bicarbonate, in the thirdexperiment, the mobile phase comprised 93.3 mM ammonium acetate and 6.7mM ammonium bicarbonate, in the fourth experiment, the mobile phasecomprised 186.7 mM ammonium acetate and 13.3 mM ammonium bicarbonate, inthe fifth experiment, the mobile phase comprised 280 mM ammonium acetateand 20 mM ammonium bicarbonate, and in the sixth experiment, the mobilephase comprised 420 mM ammonium acetate and 30 mM ammonium bicarbonate.The elution was carried out at a flow rate of 0.3 mL/min.

For analytical runs, the injection loads consisted of 10 μg of theprotein.

The use of 10 mM total salt concentration shows significant separationof the homodimer 1, bispecific antibody, and homodimer 2. At 10 mM saltconcentration, homodimer 2 had a later retention time than thebispecific antibody, which showed a later retention time thanhomodimer 1. However, at concentrations greater than 10 mM, homodimer 1had a later retention time than the bispecific antibody, which showed alower retention time than homodimer 2 (See FIG. 12 and FIG. 13). Thiseffect could be due to different type of interaction: charge, shape, orhydrophobicity of the three proteins with the size exclusionchromatography resin used. The charge on the protein at a given saltconcentration depends on their pI values (Table 2). Significantseparations were obtained either by using mobile phase with low saltconcentration of 10 mM or by using mobile phase with high saltconcentration greater than 100 mM. At lower salt concentrations,retention can be driven by charge-charge interaction. For example, basicmolecules can be separated by using mobile phase with lower saltconcentration, in the MM-SEC-MS system. At higher salt concentrations,retention is driven by hydrophobic interaction. For example, acidic orhydrophobic molecules can be separated by using mobile phase with highersalt concentration, in the MM-SEC-MS system (See FIG. 14 and FIG. 15).

An ideal SEC separation should be only based on hydrodynamic volume ofthe protein, and no other interaction should be desired between theprotein and the stationary phase. Since, silica-based column matrixmight exhibit negative charges due to silanol groups (ion-exchangecharacteristics), derivatization of the silica particle helps to reducethe silanol effect but and the same time might introduce new interactionmechanism (hydrophobicity). This can thus create SEC resins which havefunctionality, as observed with the Zenix SEC-columns. This explains thedifference in the order of elution of the proteins with differentconcentrations when carried out in a Zenix-SEC column.

TABLE 2 mAb pI MW Bispecific Antibody 7.66 145,337 Homodimer 1(Bispecific Antibody HC* 8.03 144,677 homodimer) Homodimer 2 (BispecificAntibody HC 7.28 145,998 homodimer)

Example 8. MM-SEC-MS Analysis of Deglycoslyated Mixture of BispecificAntibody, Homodimer 1, and Homodimer 2 on Waters BEH SEC Column

8.1 Preparation of Deglycoslyated Mixture of Bispecific Antibody,Homodimer 1, and Homodimer 2.

The deglycosylated mixture was prepared using the same methodology as7.1

8.2 MM-SEC-MS

The acquisition using MM-SEC-MS was performed isocratically using aWaters BEH SEC Colum on the system as described above. Elution wasmonitored by UV at 280 nm.

Six set of experiments were carried out. In the first experiment, themobile phase comprised 14 mM ammonium acetate and 1 mM ammoniumbicarbonate, in the second experiment, the mobile phase comprised 18.7mM ammonium acetate and 1.3 mM ammonium bicarbonate, in the thirdexperiment, the mobile phase comprised 28 mM ammonium acetate and 2 mMammonium bicarbonate, in the fourth experiment, the mobile phasecomprised 70 mM ammonium acetate and 5 mM ammonium bicarbonate, in thefifth experiment, the mobile phase comprised 93.3 mM ammonium acetateand 6.7 mM ammonium bicarbonate, and in the sixth experiment, the mobilephase comprised 280 mM ammonium acetate and 20 mM ammonium bicarbonate.The elution was carried out at a flow rate of 0.2 mL/min.

For analytical runs, the injection loads consisted of 10 μg of theprotein.

The use of 15 mM total salt concentration shows significant separationof the homodimer 1, bispecific antibody, and homodimer 2. At 15 mM saltconcentration, homodimer 2 had a earlier retention time than thebispecific antibody, which showed an earlier retention time thanhomodimer 1. On increasing the concentration of the mobile phase, thedifferences in the retention times reduced. Further, at saltconcentration of 300 mM of the mobile phase, homodimer 1 had a earlierretention time than the bispecific antibody, which showed a earlierretention time than homodimer 2 (See FIG. 16 and FIG. 17). As describedin FIG. 14 and FIG. 15, this effect is due to the development of anadditional interaction on the SEC column. The additional interactiondepends on the salt concentration of the mobile phase. At lowerconcentrations, the charge-charge interactions are predominant on thecolumn and determine the retention of the proteins on the column.

Example 9. MM-SEC-MS Analysis of an IgG1 Molecule and its OxidizedVariant on Waters BEH SEC Column

9.1 Preparation of an Oxidized Variant of an IgG1 Molecule—Ab1

Ab1 was treated with peptide N-glycosidase F (PNGase F; 1 IUB milliunitper 10 μg of protein) at 45° C. for 1 hour to completely remove theglycan chains from each heavy chain constant region.

9.2 MM-SEC-MS

The acquisition using MM-SEC-MS was performed isocratically using aWaters BEH SEC Colum on the system as described above. Elution wasmonitored by UV at 280 nm.

Three set of experiments were carried out. In the first experiment, themobile phase comprised 93.3 mM ammonium acetate and 6.7 mM ammoniumbicarbonate, in the second experiment, the mobile phase comprised 140 mMammonium acetate and 10 mM ammonium bicarbonate, and in the thirdexperiment, the sixth experiment, the mobile phase comprised 280 mMammonium acetate and 20 mM ammonium bicarbonate. The elution was carriedout at a flow rate of 0.2 mL/min.

For analytical runs, the injection loads consisted of 10 μg of theprotein.

There is a significant separation of the antibody Ab1 and its oxidizedvariant on MM-SEC-MS system on using 100 mM, 150 mM, and 300 mM saltconcentration of the mobile phase (See FIG. 18, top panel). The pI ofthe IgG1 antibody Ab1 is 8.65. For IgG1 molecules with higher PI, chargeinteraction plays a more dominant role compared to IgG4 molecules, whichhave low pI values.

Example 10. MM-SEC-MS Analysis of an IgG1 Molecule on Waters BEH SECColumn

10.1 Preparation of the IgG1 Molecule—Ab2

Ab2 was treated with peptide N-glycosidase F (PNGase F; 1 IUB milliunitper 10 μg of protein) at 45° C. for 1 hour to completely remove theglycan chains from each heavy chain constant region.

10.2 MM-SEC-MS

The acquisition using MM-SEC-MS was performed isocratically using aWaters BEH SEC Colum on the system as described in Example 8.2.Comparing the retention times of the two IgG1 molecules—Ab1 and Ab2, itwas observed that the Ab2 molecule had lower retention times (See FIG.18, bottom panel).

This can be explained due to hydrophobicity difference between Ab1 andAb2. For more hydrophobic molecules, the “salting out” effect starts tooccur at lower salt concentration compared to the less hydrophobicmolecules. This point is also referred to as the transition point (SeeFIG. 19).

Example 11. Quantification of Homodimer Impurities in the BispecificAntibody Using MM-SEC-MS

The standards were generated using the methodology illustrated in FIG.20.

The acquisition using MM-SEC-MS was performed isocratically using aZenix SEC-300 MK column (4.6×300 nm, 3 μm) Column on the system asdescribed. The mobile phases with 300 mM salt concentration and 70 mMsalt concentration were used to elute the proteins. For both theconcentrations in intact as well as subunit level, higher detectionusing MM-SEC-MS was observed in samples with higher amount of homodimers(See FIG. 21 and FIG. 22). At 70 mM salt concentration for mobile phase,an additional Fc impurity of the bispecific antibody was also detected.

At intact level, the plot of homodimer 1/bispecific antibody theoreticalvs. homodimer 1/bispecific antibody detected and of homodimer2/bispecific antibody theoretical vs. homodimer 2/bispecific antibodydetected showed a good linearity for quantification of homodimerspresent from 0.1% to 50% (See FIG. 23).

At subunit level, the plot of homodimer 1/bispecific antibodytheoretical vs. homodimer 1/bispecific antibody detected and ofhomodimer 2/bispecific antibody theoretical vs. homodimer 2/bispecificantibody detected showed a good linearity for quantification ofhomodimers present from 0.1% to 50% (See FIG. 24). Compared to theintact level, better accuracy was obtained at the subunit level.

Example 12. Mixed-Mode SEC Separation of Bispecific and HomodimerAntibodies for Native MS Detection

Four bsAb molecules with different pI values and hydrophobicity (Table3) were mixed with their corresponding homodimer antibodies and used asthe testing standards. Each bsAb molecule (HH*L2) contains two identicallight chains (LC) and two different heavy chains (HC and HC*), whereaseach homodimer antibody (H2L2 or H*2L2) contains two identical lightchains and two identical heavy chains (HC or HC*).

TABLE 3 Hydrophobicity (apparent HIC pI retention factor*; min) bsAb2mixture bsAb2 (HH*L2) 6.5 5.2 HC homodimer (H2L2) 6.1 4.3 HC* homodimer(H*2L2) 7.2 6.1 bsAb3 mixture bsAb3 (HH*L2) 7.4 4.8 HC homodimer (H2L2)6.6 4.2 HC* homodimer (H*2L2) 8.3 5.6 bsAb4 mixture bsAb4 (HH*L2) 7.77.0 HC homodimer (H2L2) 7.3 8.4 HC* homodimer (H*2L2) 8.0 5.7 bsAb5mixture bsAb5 (HH*L2) 8.1 6.3 HC homodimer (H2L2) 7.4 5.2 HC* homodimer(H*2L2) 8.5 7.7 *Apparent HIC retention factor was calculated based onthe retention time of the protein molecule analyzed by HIC. A YMC BioProHIC BF column (4 μm, 100 mm × 4.6 mm) was applied with mobile phase “A”of 3.3M ammonium buffer and mobile phase B of water. A gradient wasperformed from 100% to 97% A in 18 min at a flow rate of 0.4 mL/min. Theapparent HIC retention factor was calculated by normalizing itsretention time over 18 min to a scale of 10.

The four bsAb mixtures were then analyzed on the Waters BEH column usingthree different salt concentrations (75 mM, 150 mM, and 300 mM),followed by online native MS detection. The resulting base peakchromatograms (BPCs) are shown in the left panels of FIG. 25. Consistentwith the observations from the previous study, as the salt concentrationincreased, mAb molecules with different pI values exhibited differenttrends in retention times. Taking advantage of the different retentionbehavior of each antibody at varying salt concentrations, we thereforeexplored the possibility of separating the homodimers from the bsAb bymodulating the salt concentrations. For example, in the bsAb2 mixture,as the salt concentration decreased from 300 mM to 75 mM, the two acidicmolecules, H2L2 homodimer (pI=6.1) and HH*L2 bsAb (pI=6.5) both elutedearlier, likely due to reduced hydrophobic and low electrostaticinteractions at low salt concentration. In contrast, the neutralmolecule, H*2L2 homodimer (pI=7.2), remained almost unchanged inretention time as the salt concentration was modulated, presumablybecause the reduced hydrophobic interaction was counteracted by theenhanced electrostatic interaction at low salt concentration. Inaddition, as the H2L2 homodimer exhibited a more significant decrease inretention time compared to the HH*L2 bsAb (possibly due to its lower pIvalue and thus weaker electrostatic interaction), improved separationbetween the two was also achieved at a lower salt concentration. As aresult, good chromatographic separation between both homodimers andbsAb2 was achieved at 75 mM salt concentration.

Similarly, the separation between bsAb3 and its two homodimers alsoimproved significantly when the salt concentration decreased from 300 mMto 75 mM. This is because the retention times of the H2L2 homodimer(pI=6.6), the HH*L2 bsAb (pI=7.4) and the H*2L2 homodimer (pI=8.3)decreased, remained unchanged, or increased, respectively. It isnoteworthy that although baseline resolution was not achieved for eitherof the two examples, identification and quantitation of homodimersshould not be significantly impacted by the co-eluting species as theywould be for UV-based quantitation, owing to the high specificity of MSas the detector.

In the bsAb5 mixture, better separation was again achieved at 75 mM saltconcentration on the BEH column relative to the high salt condition.This is because the relatively basic molecules, HH*L2 bsAb (pI=8.1) andH*2L2 homodimer (pI=8.5), both exhibited increasingly later retentiontimes, whereas the relatively neutral H2L2 homodimer (pI=7.4) showed nochange in retention time as the salt concentration was decreased.However, despite the good chromatographic separation, this condition wasnot ideal for homodimer quantitation, as peak tailing and proteinrecovery loss started to occur for the H*2L2 homodimer at the low saltconcentration, due to its high basicity. Moreover, the bsAb4 mixturedemonstrated that improved separation could not be achieved bydecreasing the salt concentration from 300 mM to 75 mM. This is likelybecause the three molecules all have near neutral pIs (Table 3), andthus exhibit similar retention behavior with corresponding saltconcentration changes. Although further lowering the salt concentrationto enhance electrostatic interaction may improve the separation, severepeak tailing will likely occur, thus compromising the quantitation. Itis also interesting to note that the elution profile of the bsAb4mixture broadened as the salt concentration increased. At 300 mM saltconcentration, the elution order of the three molecules was determinedby XICs (data not shown) as H*2L2 homodimer, HH*L2 bsAb, and H2L2homodimer, which was consistent with their ranking in hydrophobicitydetermined by hydrophobic interaction chromatography (Table 3).Therefore, further enhancing the hydrophobic interaction by using aneven higher salt concentration will likely improve the separation ofbsAb4 mixture on this column. Unfortunately, based on our experience,salt concentrations higher than 300 mM usually creates a desolvationissue and significantly impairs native MS sensitivity.

To further examine the separation of the bsAb mixtures, while using saltconcentrations favorable for MS analysis, a Sepax Zenix SEC-300 columnwas evaluated for mixed-mode interactions. The 3 μm silica beads in thiscolumn are coated with a chemically bonded, stand-up monolayer, whichlikely contributes to the moderate hydrophobicity of this column asreported in previous studies (Yang et al, 2016, supra; Wong et al,supra; Pavon et al, supra). At 150 mM and 300 mM salt concentrations,each of the four bsAb mixtures were separated on this column forsubsequent native MS detection, and the generated BPCs are shown in theright panels of FIG. 25. As expected, the bsAb4 mixture showed improvedseparation on the Zenix column compared to the BEH column when operatedat the same salt concentrations. The elution order of the threemolecules was also consistent with their relative hydrophobicity, withthe most hydrophobic H2L2 homodimer eluting last. Note that thechromatographic resolution of the bsAb4 mixture was further improved at300 mM salt concentration compared to that at 150 mM, which is expectedas a higher salt concentration promotes hydrophobic interaction. Inaddition, the bsAb5 mixture was more effectively resolved on the Zenixcolumn compared to the BEH column, presumably due to the largedifferences in hydrophobic interaction with the column matrix betweenmixture components. In summary, by modulating the salt concentration ontwo SEC columns with different properties, we demonstrate that goodchromatographic separation can be achieved for all four bsAb mixtures atsalt concentrations favorable for subsequent native MS detection. It isalso likely that other SEC columns, not tested in this study, canfurther extend the applicability of this method, by offering novelmixed-mode interactions.

Example 13. Quantitation of Homodimer Impurities by Native MM-SEC-MS

Relative quantitation by MS-based approaches often requires awell-characterized understanding of MS response (e.g., ionizationefficiency and ion transmission efficiency) from each analyte. Becauseof the similar size, bsAb and homodimers should exhibit similar iontransmission efficiency during native MS analysis. On the other hand,ionization efficiency could be affected by both the solvent compositionat the time of elution and the presence of co-eluting species. As theMM-SEC-MS method utilizes isocratic elution, influences on ionizationdue to different solvent composition, as commonly seen in gradientelution methods (e.g., IEX-MS), can be eliminated. To evaluate theperformance of the MM-SEC-MS method to assess relative quantitation ofhomodimer impurities, a series of bsAb2 spiked-in samples containinghomodimer impurities ranging from relative abundances 0.1% to 10% wereprepared for analysis. The Waters BEH column using the 75 mM saltconcentration mobile phase was applied to achieve MM-SEC separationbetween bsAb2 and the corresponding homodimers. To assess the relativequantitation of each homodimer present within the bsAb2 samples, theXICs, based on the m/z of the four most abundant charge states of eitherhomodimer species or bsAb2, were generated and the peak areas wereintegrated and used for quantitation of the amount of each homodimer. Asshown in FIG. 26, reliable quantitation of homodimer impurities rangingfrom 0.1% to 10% can be readily achieved by this method. In addition,even at a 0.1% spiked-in level, high-quality native mass spectra of bothH2L2 and H*2L2 homodimer species can still be obtained (FIG. 27, rightpanel), leading to high-confidence identification and quantitation.

A novel MM-SEC-MS method has been developed and evaluated for highlysensitive detection and quantitation of homodimer impurities in bsAbsamples. We first investigated the mixed-mode interactions between theantibody molecule and the column matrix during SEC separation atdifferent salt concentrations. Using eight distinct antibodies ofvarying pI, it was observed that under a defined pH condition, the basicmolecules exhibited stronger electrostatic interactions with the columnmatrix compared to the acidic molecules, and such interaction can beenhanced by lowering the salt concentration. On the other hand,increasing the salt concentration during SEC separation can reduceelectrostatic interaction, while promoting hydrophobic interactionsbetween the antibody and the column matrix. These mixed-modeinteractions provide a unique opportunity for separating antibodies withsimilar hydrodynamic volume but different surface characteristics.Taking advantage of different column properties, chromatographicseparation of four bsAb mixtures was accomplished by the MM-SEC methodusing either electrostatic interaction or hydrophobic interaction, whichwas readily achieved by modulating salt concentrations. We alsodemonstrated that the achieved chromatographic separation was criticalto obtain improved detection of low-abundance homodimer impurities bysubsequent native MS analysis. In two bsAb examples, homodimerimpurities present at 0.01% (bsAb2) and 0.1% (bsAb4) were successfullydetected using this MM-SEC-MS method. To the best of our knowledge, thisnew development represents the most sensitive method in detectinghomodimer impurities in bsAb samples. Finally, using a series ofspiked-in standards, we demonstrated that the MM-SEC-MS method candeliver reliable quantitation of homodimer impurities present at varyinglevels. Owing to the high sensitivity, high-confidence identificationand quantitation can be obtained even at levels as low as 0.1%. Insummary, this newly developed MM-SEC-MS method provides a highlysensitive approach for detection and quantitation of homodimerimpurities in bsAb samples and thus can be used to support therapeuticbsAb development. Finally, application of this method might extend toother areas, such as characterization of a mixture of antibodies presentin co-formulated therapeutics.

What is claimed is:
 1. A method for quantifying an impurity in a sample,said method comprising: contacting said sample to a chromatographicsystem having a mixed-mode size exclusion chromatography resin with anadditional functionality; washing said mixed-mode size exclusionchromatography resin using a mobile phase to provide an eluent includingthe impurity; and quantifying an amount of the impurity in said eluentusing a mass spectrometer.
 2. The method of claim 1, wherein the mobilephase used to elute the impurity has ammonium acetate, ammoniumbicarbonate, or ammonium formate, or combinations thereof.
 3. The methodof claim 1, wherein the mobile phase used to elute the impurity has atotal concentration of less than about 600 mM of ammonium acetate andammonium bicarbonate.
 4. The method of claim 1, wherein the mobile phaseused to elute the impurity has a flow rate of about 0.2 ml/min-about 0.4ml/min.
 5. The method of claim 1, wherein the amount of the sampleloaded onto the mixed-mode size exclusion chromatography resin is about10 μg to about 100 μg.
 6. The method of claim 1, wherein the impurity isa product-related impurity.
 7. The method of claim 1, wherein theimpurity is a homodimer.
 8. The method of claim 1, wherein the sampleincludes the impurity and at least one target protein that are separatedwhen eluted.
 9. The method of claim 8, wherein the target protein is anantibody.
 10. The method of claim 9, wherein the antibody is abispecific antibody.
 11. The method of claim 8, wherein the targetprotein is a therapeutic antibody.
 12. The method of claim 1, whereinthe mass spectrometer is coupled to the chromatographic system.
 13. Themethod of claim 1, wherein the additional functionality is a hydrophobicinteraction functionality.
 14. The method of claim 1, wherein theadditional functionality is a charge-charge interaction functionality.15. The method of claim 1, wherein the mass spectrometer is a nativemass spectrometer.
 16. A method for detecting an impurity in a sample,said method comprising: contacting said sample to a chromatographicsystem having a mixed-mode size exclusion chromatography resin with anadditional functionality; washing said mixed-mode size exclusionchromatography resin using a mobile phase to provide an eluent includingthe impurity; and detecting the impurity in said eluent using a massspectrometer.
 17. A mixed-mode chromatography system comprising: achromatographic column having a mixed-mode size exclusion chromatographyresin with an additional functionality, wherein the column is capable ofreceiving a mobile phase and a sample having a target protein, and amass spectrometer coupled to said chromatographic column for detectingthe target protein.
 18. The mixed-mode chromatography system of claim17, wherein the additional functionality is a hydrophobic interactionfunctionality.
 19. The mixed-mode chromatography system of claim 17,wherein the additional functionality is a charge-charge interactionfunctionality.
 20. The mixed-mode chromatography system of claim 17,wherein the system is further capable of quantifying a target protein.